U.S. patent number 5,733,719 [Application Number 08/445,217] was granted by the patent office on 1998-03-31 for method of making an assay compound.
This patent grant is currently assigned to Coulter Corporation. Invention is credited to James H. Carter, Gerald E. Jaffe, Frank J. Lucas.
United States Patent |
5,733,719 |
Jaffe , et al. |
March 31, 1998 |
Method of making an assay compound
Abstract
An assay compound or a salt thereof for assaying the activity of
an enzyme inside a metabolically active whole cell is disclosed.
The assay compound includes a leaving group and an indicator group.
The leaving group is selected from the group comprising amino
acids, peptides, saccharides, sulfates, phosphates, esters,
phosphate esters, nucleotides, polynucleotides, nucleic acids,
pyrimidines, purines, nucleosides, lipids and mixtures thereof. The
indicator group is selected from compounds which have a first state
when joined to the leaving group, and a second state when the
leaving group is cleaved from the indicator group by the enzyme.
Preferably, the indicator compounds are rhodamine 110, rhodol, and
fluorescein and analogs of these compounds. A method of
synthesizing the compound as well as methods of using these
compounds to measure enzyme activity are also disclosed.
Inventors: |
Jaffe; Gerald E. (Pembroke
Pines, FL), Lucas; Frank J. (Boca Raton, FL), Carter;
James H. (Plantation, FL) |
Assignee: |
Coulter Corporation (Miami,
FL)
|
Family
ID: |
23768038 |
Appl.
No.: |
08/445,217 |
Filed: |
May 18, 1995 |
Current U.S.
Class: |
435/4; 435/29;
435/968 |
Current CPC
Class: |
C12Q
1/34 (20130101); G01N 33/574 (20130101); Y10S
435/968 (20130101) |
Current International
Class: |
C12Q
1/34 (20060101); G01N 33/574 (20060101); C12Q
001/00 (); C12Q 001/02 () |
Field of
Search: |
;435/4,18,29,808,968 |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
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1945663 |
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Mar 1971 |
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DE |
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9310461 |
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May 1993 |
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WO |
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Other References
Mitchell, G., Novel Rhodamine Tripeptide Substrate for Manual and
Automated . . . Time Test, Thrombosis Research, vol. 40, 339-349,
1985. .
Hegazi, F., Factors Affecting the Caseinolytic Activity of
Lactobacillus plantarum, Die Nahrung, vol. 31, No. 3, 199-206,
1987. .
Watson, Cytometry, vol. 1, No. 2, pp. 143-151 (1980). .
Leytus et al, Biochem. J., vol. 209, pp. 299-307 (1983). .
Leytus et al, Biochimica et Biophysica Acta, vol. 788, pp. 74-86
(1984). .
Melhado et al, J. Am. Chem. Soc., vol. 104, pp. 7299-7306 (1982).
.
Leytus et al, Biochem. J., vol. 215, pp. 253-260 (1983). .
Kanaoka et al, Chem. Pharm. Bull, vol. 25, No. 2, pp. 362-363
(1977). .
Morita et al, J. Biochem., vol. 82, pp. 1495-1498 (1977). .
Mononen et al, Clinical Chemistry, vol. 40, No. 3, pp. 385-388
(1994). .
Livingston et al, Biochemistry, vol. 20, No. 15, pp. 4298-4306
(1981). .
Rothe et al, Biol. Chem. Hoppe-Seyler, vol. 373, pp. 547-554
(1992). .
Mangel et al, Nature, vol. 361, pp. 274-275 (Jan. 21, 1993). .
Huang et al, The Journal of Histochemistry and Cytochemistry, vol.
41, No. 2, pp. 313-317 (1993). .
Lottenberg et al, Methods in Enzymology, vol. 80, pp. 341-361
(1981). .
Saifuku et al, Clinica Chimica Acta, vol. 84, No. 1/2, pp. 85-91
(1978). .
Dive et al, Cytometry, vol. 8, pp. 552-561 (1987). .
BioProbes 21--New Products and Applications from Molecular Probes,
Inc., cover page, contents page and pp. 18-21 (Nov. 1994). .
Duffy et al, Clinical Chemistry, vol. 38, No. 10, pp. 2114-2116
(1992). .
Valet et al, Ann. NY Acad. Sci., vol. 677, pp. 233-251 (1993).
.
Cox et al, Cytometry, vol. 8, pp. 267-272 (1987). .
Hjorth, Brain Topography, vol. 2, Nos. 1/2, pp. 57-61 (1989). .
Windham et al, Journal of Computer Assisted Tomography, vol. 12,
No. 1, pp. 1-9 (1988)..
|
Primary Examiner: Gitomer; Ralph
Attorney, Agent or Firm: Alter; Mitchell E.
Claims
We claim:
1. A method to make an assay compound for assaying the activity of
an enzyme inside a metabolically active whole cell, said assay
compound comprising an indicator group and a leaving group, said
leaving group being selected for cleavage by said enzyme,
comprising:
a. reacting a compound containing a leaving group selected from the
group consisting of amino acids, peptides, phosphate esters,
saccharides, esters, nucleotides, polynucleotides, nucleic acids,
pyrimidines, purines, nucleosides, lipids and mixtures thereof and
a blocking group, with an agent to form an intermediate complex
containing a leaving group and a blocking group;
b. reacting the intermediate complex with a compound containing an
indicator group to form a reaction product, wherein said indicator
group is at least one selected from the group consisting of
rhodamine 110, rhodol, fluorescein and derivatives thereof;
c. separating the reaction product from side reaction products,
by-products and starting materials;
d. removing the blocking group from the reaction product to obtain
an assay compound having an indicator group and leaving group;
and
e. purifying the assay compound so that fluorescence of impurities
in such assay compound is less than autofluorescence of the
metabolically active cell.
2. The method of claim 1, wherein the blocking group is at least
one selected from the group consisting of formyl, acetyl,
triftuoroacetyl, benzyloxycarbonyl, phthaloyl, benzoyl,
acetoacetyl, chloroacetyl, phenoxycarbonyl, carbobenzoxy,
substituted benzyloxycarbonyl, tertbutyloxycarbonyl,
isopropyloxycarbonyl, allyloxycarbonyl, methoxysuccinyl, succinyl,
2,4-dinitrophenyl, dansyl, p-methoxybenzenesulfonyl, and
phenylthio.
3. The method of claim 1 wherein the blocking group is at least one
selected from the group consisting of acetyl, benzyloxycarbonyl,
tertbutoxycarbonyl(t-BOC), and
9-fluorenylmethyloxycarbonyl(FMOC).
4. The method of claim 1, wherein said derivatives comprise
4'(5')aminorhodamine 110, 4'(5')carboxyrhodamine 110,
4'(5')chlororhodamine 110, 4'(5')methylrhodamine 110,
4'(5')sulforhodamine 110, rhodol, 4'(5')aminorhodol,
4'(5')carboxyrhodol, 4'(5')chlororhodol, 4'(5')methylrhodol,
4'(5')sulforhodol, fluorescein, 4'(5')aminofluorescein,
4'(5')carboxyfluorescein, 4'(5')chlorofluorescein,
4'(5')methylfluorescein, and 4'(5')sulfofluorescein.
5. The method of claim 1, wherein said agent utilized to form an
intermediate complex is selected from the group consisting of a
substituted carbodiimide, benzotriazolyl-N-oxy-tris(dimethylamino)
phosphonium hexafluorophosphate and 1-hydroxybenzo-triazole.
6. A method to make an assay compound for assaying the activity of
an enzyme inside a metabolically active whole cell, said assay
compound comprising an indicator group and a leaving group, said
leaving group being selected for cleavage by said enzyme,
comprising:
a. reacting a compound containing a leaving group selected from the
group consisting of amino acids, peptides, sulfates, phosphates,
esters, phosphate esters, nucleotides, polynudeotides, nucleic
acids, pyrimidines, purines, nucleosides, lipids and mixtures
thereof and a blocking group, with an agent to form an intermediate
complex containing a leaving group and a blocking group;
b. reacting the intermediate complex with a compound containing an
indicator group to form a reaction product, wherein said indicator
group is at least one selected from the group consisting of
rhodamine 110, rhodol, fluorescein and derivatives thereof;
c. separating the reaction product from side reaction products,
by-products and starting materials;
d. removing the blocking group from the reaction product to obtain
an intermediate compound having an indicator group and leaving
group;
e. reacting the intermediate compound having an indicator group and
leaving group with an acid or a base to form a physiologically
acceptable salt of said assay compound for assaying the activity of
an enzyme inside a metabolically active which cell; and
f. purifying the physiologically acceptable salt of said assay
compound so that fluorescence of impurities in such assay compound
is less than autofluorescence of the metabolically active cell.
7. The method of claim 6, wherein the blocking group is at least
one selected from the group consisting of formyl, acetyl,
trifluoroacetyl, benzyloxycarbonyl, phthaloyl, benzoyl,
acetoacetyl, chloroacetyl, phenoxycarbonyl, carbobenzoxy,
substituted benzyloxycarbonyl, tertbutyloxycarbonyl,
isopropyloxycarbonyl, allyloxycarbonyl, methoxysuccinyl, succinyl,
2,4-dinitrophenyl, dansyl, p-methoxybenzenesulfonyl, and
phenylthio.
8. The method of claim 7, wherein the blocking group is at least
one selected from the group consisting of tertbutyloxy-carbonyl,
9-fluorenylmethyloxycarbonyl, and acetyl.
9. The method of claim 7, wherein said derivative comprises 4'(5')
aminorhodamine 110, 4'(5')carboxyrhodamine 110,
4'(5')chlororhodamine 110, 4'(5')methylrhodamine 110,
4'(5')sulforhodamine 110, rhodol, 4'(5')aminorhodol,
4'(5')carboxyrhodol, 4'(5')chlororhodol, 4'(5')methylrhodol,
4'(5')sulforhodol, fluorescein, 4'(5')aminofluorescein,
4'(5')carboxyfluorescein, 4'(5')chlorofluorescein,
4'(5')methyl-fluorescein, and 4'(5')sulfofluorescein.
10. The method of claim 6, wherein said salt is an acid salt
selected from the group consisting of hydrochloric, maleic, acetic,
trifluoroacetic, tartaric acid, citric, succinic, and
p-toluenesulfonic acid or a base salt selected from the group
consisting of ammonia and organic bases.
Description
BACKGROUND OF THE INVENTION
1. Field of the Invention
This invention relates generally to cytoenzymology, and more
particularly reagents for use in cytoenzymology as well as
production and use of these reagents.
2. Description of the Background Art
Cytoenzymology is the study of enzymes as they function on and
within cells. Previously, the study of enzymatic activity within
cells has been pursued primarily by two indirect methods. According
to a first method, the cell membrane is broken to create a cytosol
of cellular components including the enzyme-which is the object of
study. Various tests are then performed to determine the activity
of the enzyme, which tests can be performed on the cytosol or on
the purified enzyme. According to a second method, the enzyme
activity is determined from the study of extra-cellular events,
such as the presence or lack of the products of enzyme
activity.
According to the first method, various tests are performed to
determine enzyme activity in the cytosol. One such test is to
provide a substrate that is recognized by the enzyme, with a
fluorescent compound which will undergo a detectable change when
the substrate, or "leaving group", is cleaved from the compound by
the enzyme. Mangel et al., U.S. Pat. Nos. 4,557,862 and 4,640,893,
disclose rhodamine 110-based derivatives as fluorogenic substrates
for proteinases. These compounds have the general formula:
where the peptide includes known amino acids or amino acid
derivatives, and "Cbz" refers to the blocking group
benzyloxycarbonyl. When the amino groups of rhodamine 110 are
blocked the compound is "quenched", and is relatively colorless and
non-fluorescent. Cleavage of one of the peptides from the
non-fluorescent bisamide substrate results in a 3500-fold increase
in fluorescence intensity.
The rhodamine 110 substrates of Mangel et al. have been utilized to
conduct cytoenzymological studies. G. Rothe et al., Biol. Chem.
Hoppe-Seyler, 373, 544-547 (1992) describe the analysis of
proteinase activities using the substituted peptide-rhodamine 110
derivatives of Mangel et al. Moreover, G. Valet et al, Ann NY Acad
Sci, 667, 233-251 (1993), disclose the study of white cell and
thrombocyte disorders with the rhodamine 110 derivatives of Mangel
et al. The methods of Rothe and Valet have been used to conduct
cytoenzymological studies on the activity of enzymes with cells,
but the compounds utilized by Rothe and Valet are not suitable for
the study of the activity of intracellular enzymes in vital cells.
The Mangel et al. compounds cannot be efficiently solubilized and
transmitted through the cell membrane in a manner which will
produce a reliable assay. In addition, the Cbz group in the Mangel
et al. compound is not recognized by the enzyme's active sites.
Further, Mangel, et al., disclose the removal of the carbobenzyloxy
group by treating the blocked peptide-indicator compound with 30%
hydrobromide acid in acetic acid. However, the bromide salt is
lethal to the cell and does not permit an assay for a metabolically
active cell.
I. Mononen, et al., Clin. Chem., 40 (3), 385-388 (1994), describe
the enzymatic diagnosis of aspartylglycosaminuria by the
fluorometric assay of glycosylasparaginase in serum, plasma, and
lymphocytes. The study was conducted on cytosols, and not whole
cells, and utilized an asparagine-substituted
7-amino-4-methylcoumarin.
Dead or metabolically inactive cells can have as little as
approximately one-quarter the enzymatic activity of living cells,
Watson, J., "Enzyme Kinetic Studies in Cell Population Using
Fluorogenic Substrates and Flow Cytometric Techniques", Cytometry,
1(2), p. 143 (1980). Further, because enzymes are frequently bound
in highly organized enzyme pathways, the disruption and death of
the cell can greatly affect enzyme activity. Current assays
therefore have limited utility for determining enzyme activity in a
living or metabolically active whole cell.
U.S. Pat. No. 5,070,012 to Nolan et al., describes a method of
monitoring cells and trans-acting transcription elements. This
method, however, is not designed for the monitoring of enzymes
which are endogenous to the cell being tested. Rather, in this
method a hypotonic solution is used to increase the permeability of
the cell membrane thereby allowing an exogenous enzyme and other
reagents (including a fluorogenic substrate) to be introduced into
the cell. However, these severe hypotonic conditions significantly
alter the normal state of the cell. The fluorogenic substrate
described in this patent (fluoroscein digalactopyranoside) contains
significant amounts of fluorescent impurities and must be bleached
with a laser prior to use.
SUMMARY OF THE INVENTION
The present invention relates to an assay reagent for determining
the activity of an enzyme in a metabolically active whole cell,
said assay reagent comprising at least one water soluble assay
compound having the ability to pass through a cell membrane or a
water soluble physiologically acceptable salt thereof having the
ability to pass through a cell membrane, said assay compound having
a leaving group selected for cleavage by an enzyme to be analyzed
and a fluorogenic indicator group being selected for its ability to
have a non-fluorescent first state when joined to the leaving
group, and a fluorescent second state excitable at a wavelength
above 450 nm when the leaving group is cleaved from the indicator
group by the enzyme, said assay reagent having a fluorescence less
than the auto-fluorescence of a metabolically active cell and being
stable for a minimum of 30 days when stored at wherein said
stability is defined as the compound having an increase in
background fluorescence of .ltoreq.10%.
The present invention also relates to an assay reagent for
determining the activity of an enzyme in a metabolically active
whole cell, said assay reagent comprising at least one water
soluble salt of an assay compound having the ability to pass
through a cell membrane, said assay compound having a leaving group
selected for cleavage by an enzyme to be analyzed and a fluorogenic
indicator group being selected for its ability to have a first
non-fluorescent state when joined to the leaving group, and a
second fluorescent state excitable at a wavelength above 450 nm
when the leaving group is cleaved from the indicator group by the
enzyme, and said assay reagent having a fluorescence less than the
auto-fluorescence of a metabolically active cell.
The present invention also relates to an assay reagent composition
for determining the activity of an enzyme in a metabolically active
whole cell, said assay reagent comprising at least one water
soluble assay compound having the ability to pass through a cell
membrane or a water soluble physiologically acceptable salt thereof
having the ability to pass through a cell membrane, said assay
compound having a leaving group selected for cleavage by an enzyme
to be analyzed and a fluorogenic indicator group being selected for
its ability to have a non-fluorescent first state when joined to
the leaving group, and a fluorescent second state excitable at a
wavelength above 450 nm when the leaving group is cleaved from the
indicator group by the enzyme, and at least one additive selected
from the group consisting of a buffer, an enzyme cofactor, an
enzyme modulator, an enzyme inhibitor, an enzyme activator, a
solubilizing component for said assay reagent, and a retention
component for said assay reagent or products thereof, said assay
reagent having a fluorescence less than the auto-fluorescence of a
metabolically active cell.
This invention also relates to a method to produce an assay reagent
for determining the activity of an enzyme in a metabolically active
whole cell, in which the cell is contacted with the assay reagent.
In a broad aspect, the invention relates to a method to make an
assay compound for assaying the activity of an enzyme inside a
metabolically active whole cell, said assay compound comprising an
indicator group and a leaving group, said leaving group being
selected for cleavage by said enzyme, comprising reacting a
compound containing a leaving group selected from the group
consisting of amino acids, peptides, saccharides, sulfates,
phosphates, esters, phosphate esters, nucleotides, polynucleotides,
nucleic acids, pyrimidines, purines, nucleosides, lipids and
mixtures thereof and a blocking group, with an agent to form an
intermediate complex containing a leaving group and a blocking
group, reacting the intermediate complex with a compound containing
an indicator group to form a reaction product; separating the
reaction product from side reaction products, by-products and
starting materials, removing blocking groups from the reaction
product to obtain an assay compound having an indicator group and
leaving group, optionally reacting the intermediate compound having
an indicator group and leaving group with an acid or base to form a
physiologically acceptable salt of said assay compound for assaying
the activity of an enzyme inside a metabolically active whole cell,
and purifying the assay compound or the physiologically acceptable
salt thereof.
More specifically, the present invention is further related to a
method for making an assay compound for assaying the activity of an
enzyme inside a metabolically active whole cell, said assay
compound comprising an indicator group and a leaving group, said
leaving group being selected for cleavage by said enzyme,
comprising reacting a compound containing a leaving group selected
from the group consisting of amino acids, peptides, saccharides,
esters, nucleotides, lipids and mixtures thereof, and a blocking
group with an agent to form an intermediate complex containing a
leaving group and a blocking group, reacting the intermediate
complex with a compound containing an indicator group to form a
reaction product, separating the reaction product from side
reaction products, by-products and starting materials, removing the
blocking group from the reaction product to obtain an assay
compound having an indicator group and leaving group, and purifying
the assay compound.
In another embodiment, the present invention is further related to
a method for making an assay compound in a salt form for assaying
the activity of an enzyme inside a metabolically active whole cell,
said assay compound comprising an indicator group and a leaving
group, said leaving group being selected for cleavage by said
enzyme, comprising reacting a compound containing a leaving group
selected from the group consisting of amino acids, peptides,
phosphates, sulfates, esters, nucleotides and mixtures thereof, and
a blocking group with an agent to for man intermediate complex
containing a leaving group and a blocking group, reacting the
intermediate complex with a compound containing an indicator group
to form a reaction product, separating the reaction product from
side reaction products, by-products and starting materials,
removing the blocking group from the reaction product to obtain an
assay compound having an indicator group and leaving group,
reacting the intermediate compound having an indicator group and
leaving group with an acid or a base to form a physiologically
acceptable salt of said assay compound for assaying the activity of
an enzyme inside a metabolically active whole cell, and purifying
the physiologically acceptable salt of said assay compound.
The assay reagent has at least one assay compound having an
indicator group and a leaving group. The leaving group is selected
for cleavage by the enzyme to be assayed. The indicator group is in
a first state when joined to the leaving group (e.g. the indicator
is non-fluorescent), and is in a second state when the leaving
group is cleaved from the indicator group by the enzyme (e.g. the
indicator group is fluorescent).
The present invention also relates to a method for determining the
activity of an endogenous enzyme in a metabolically active whole
cell, comprising contacting a metabolically active whole cell with
an assay reagent under conditions which allow said assay reagent to
pass into said metabolically active whole cell, said assay reagent
having at least one assay compound having the ability to pass
through a cell membrane or a physiologically acceptable salt
thereof having the ability to pass through a cell membrane, said
assay compound comprising a fluorogenic indicator group and a
leaving group, said leaving group being selected for cleavage by
said enzyme, said indicator group being in a non-fluorescent first
state when joined to said leaving group, and being in a fluorescent
second state excitable at a wavelength above 450 nm when said
leaving group is cleaved from said indicator group by said enzyme
for a period of time sufficient for said assay reagent to be
transferred into said cell and for said leaving group to be cleaved
inside said cell from said indicator group by said enzyme, exposing
said cell to light having a wavelength above 450 nm, and measuring
fluorescence of said cell.
The present invention also relates to a method for detecting an
abnormality in the activity of an enzyme in a metabolically active
whole cell, comprising (a) contacting a reference, metabolically
active whole cell having a normally functioning enzyme with a
medium containing an assay reagent, said assay reagent having at
least one water soluble assay compound having the ability to pass
through a cell membrane or a water soluble physiologically
acceptable salt thereof having the ability to pass through a cell
membrane, said assay compound comprising a fluorogenic indicator
group and a leaving group, said leaving group being selected for
cleavage by said enzyme, said indicator group being in a
non-fluorescent first state when bonded to said leaving group, and
being in a fluorescent second state excitable at a wavelength above
450 nm when said leaving group is cleaved from said indicator group
by said enzyme, for a period of time sufficient for said assay
compound to be transferred into said cell and for said leaving
group to be cleaved inside said cell from said indicator group by
said enzyme, (b) sensing for said fluorescent second state of said
indicator group for the reference, metabolically active whole cell
to produce reference results, (c) contacting a test, metabolically
active whole cell with said medium for said period of time, (d)
sensing for said fluorescent second state of said indicator group
for the test, metabolically active whole cell to produce test
results, and (e) comparing the reference results of reference test,
metabolically active whole cell in said step (b) with the test
results obtained from said test metabolically active whole cell in
said step (d).
The present invention also relates to a method of performing an
assay for detecting the presence of a disease comprising (a)
contacting a test, metabolically active whole cell with an assay
reagent, said assay reagent containing at least one water soluble
assay compound or water soluble physiologically acceptable salt
thereof having a fluorogenic indicator group and a leaving group,
said leaving group being selected for cleavage by a enzyme the
activity of which changes with the presence of the disease, said
indicator group being in a non-fluorescent first state when bonded
to said leaving group, and being in a fluorescent second state
excitable at a wavelength above 450 nm when said leaving group is
cleaved from said indicator group by said enzyme for a period of
time at least sufficient for said assay compound to be transferred
into said cell and for said leaving group to be cleaved inside said
cell from said indicator group by said enzyme, (b) sensing for said
fluorescent second state of the indicator group for the test,
metabolically active whole cell to produce test results, and (c)
comparing the test results of said test metabolically active whole
cell with reference results obtained from at least one of a
diseased reference cell and a non-diseased reference cell.
The present invention also relates to a method for detecting an
abnormality in the activity of an enzyme in a metabolically active
whole cell, comprising (a) contacting a plurality of reference,
metabolically active whole cells, each having at least one normally
functioning enzyme with a medium containing an assay reagent, said
assay reagent having at least one water soluble assay compound
having the ability to pass through a cell membrane or a water
soluble physiologically acceptable salt thereof having the ability
to pass through a cell membrane, said assay compound comprising a
fluorogenic indicator group and a leaving group, said leaving group
being selected for cleavage by one of said at least one normally
functioning enzymes, said indicator group being in a
non-fluorescent first state when bonded to said leaving group, and
being in a fluorescent second state excitable at a wavelength above
450 nm when said leaving group is cleaved from said indicator group
by the one of said at least one normally functioning enzyme, for a
period of time sufficient for said assay compound to be transferred
into each of said plurality of reference, metabolically active
whole cells for each of the at least one normally functioning
enzymes for each of said plurality of reference, metabolically
active whole cells to produce a matrix of reference results and for
said leaving group to be cleaved inside of each of said plurality
of reference, metabolically active whole cells from said indicator
group by the one of said at least one normally functioning enzymes,
(b) sensing for said fluorescent second state, of said indicator
group for each of the at least one normally functioning enzymes for
each said plurality of reference, metabolically active whole cells
to produce a matrix of reference results, (c) contacting a
plurality of test, metabolically active whole cells, each having at
least one normally functioning enzyme with said medium for said
period of time, (d) sensing for said fluorescent second state of
said indicator group for each of the at least one normal
functioning enzyme for each of said plurality of test,
metabolically active whole cells to produce a matrix of test
results, and (e) comparing the matrix of test results of said
plurality of test, metabolically active whole cells in said step
(d) with the matrix of reference results obtained from said
plurality of reference, metabolically active whole cells in said
step (b).
The present invention also relates to a method of performing at
least one or more assays for detecting the presence of a disease
comprising (a) contacting at least one or more test, metabolically
active whole cells with one or more assay reagents, said assay
reagent containing at least one water soluble assay compound or
water soluble physiologically acceptable salt thereof having a
fluorogenic indicator group and a leaving group, said leaving group
being selected for cleavage by one of said at least one normally
functioning enzyme the activity of which changes with the presence
of the disease, said indicator group being in a non-fluorescent
first state when bonded to said leaving group, and being in a
fluorescent second state excitable at a wavelength above 450 nm
when said leaving group is cleaved from said indicator group by the
one of said at least one normally functioning enzyme for a period
of time at least sufficient for said assay compound to be
transferred into said cell and for said leaving group to be cleaved
inside said cell from said indicator group by said enzyme, (b)
sensing for said fluorescent second state of the indicator group
for the test, metabolically active whole cell to produce a matrix
of test results, and (c) comparing the matrix of test results of
said test metabolically active whole cell with a matrix of
reference results obtained from at least one of a diseased
reference cell and a non-diseased reference cell.
The cell is contacted with the assay compound for a period of time
sufficient for the assay reagent to be transferred into the cell
and for the leaving group to be cleaved from the indicator group by
the enzyme. The assay compound is capable of or enabled to pass
through the membrane of the cell so that the enzyme, if present and
active, can cleave the leaving group thereby forming the indicator
compound which can be sensed from outside the cell.
The cell is then sensed for the first state or second state or both
first and second states of the indicator group.
BRIEF DESCRIPTION OF THE DRAWINGS
The file of this patent contains at least one drawing executed in
color. Copies of this patent with color drawings will be provided
by the Patent and Trademark Office upon request and payment of the
necessary fee.
There are shown in the drawings embodiments which are presently
preferred, it being understood, however, that the invention is not
limited to the precise instrumentalities and arrangements shown,
wherein:
FIGS. 1A, 1B, 1C and 1D are flow charts of four assay protocols
according to the invention;
FIGS. 2A, 2B, 2C and 2D are charts illustrating the use of salts to
enhance specificity;
FIGS. 3A, 3B, 3C and 3D are graphs illustrating the use of
inhibitors in the reagent formula;
FIGS. 4A and 4B are photomicrographs of normal Ficoll prepared
lymphocytes and acute lympholytic Ficoll prepared lymphocytes,
respectively, which illustrate use of an assay according to the
invention to determine immune competence and the difference in
enzyme activity between normal lymphocytes and acute lymphocytic
leukemia lymphocytes.
FIGS. 5A and 5B are color photomicrographs which illustrate use of
an assay according to the invention to provide an indication of
leukemia;
FIGS. 6A and 6B are color photomicrographs which illustrate use of
an assay to provide an indication of sepsis;
FIG. 7 is a color photomicrograph which illustrates the use of an
assay to provide an indication of the metastatic potential of
tumors;
FIGS. 8A and 8B are color photomicrographs which illustrate the use
of assays to monitor drug treatment;
FIG. 9 is a color photomicrograph which illustrates the use of
assays to provide an indication of macrophage. activation;
FIGS. 10A and 10B are graphs illustrating the storage stability of
a monopeptide-TFA salt derivative of rhodamine 110;
FIGS. 11A, 11B, 11C, 11D, 11E and 11F are graphs illustrating the
storage stability of TFA salts of dipeptide derivatives of
rhodamine 110;
FIGS. 12A, 12B, 12C and 12D are graphs illustrating the storage
stability of acetate and tartrate salts of the Leu-Gly peptide
derivative of rhodamine 110;
FIGS. 13A and 13B are graphs illustrating the storage stability of
free-amine peptide derivatives of rhodamine 110;
FIG. 14A illustrates reducing a full covariance data matrix to a
reduced covariance data matrix of strongly contributing factors, by
eigenvector analysis;
FIG. 14B illustrates the prediction of disease probabilities from
the reduced covariance data matrix using Non-Negative Least Squares
(NNLS) analysis;
FIG. 15 illustrates a full covariance data matrix being fed to a
Neural Network to predict disease probabilities;
FIG. 16 illustrates a comparison of disease probabilities by
performing 1) NNLS analysis on the full covariance data matrix, 2)
NNLS analysis on a reduced covariance data matrix defined by
eigenvectors 1 and 2, including 11 substrates, 3) NNLS analysis on
a reduced covariance data matrix defined by eigenvector 1 above,
including 6 substrates, and 4) squared deviation from the mean
analysis on 6 substrates;
FIGS. 17A-17C are graphs of the ratio of a disease to the mean of
the normal of all patients with the disease; and
FIGS. 18A-18F illustrate a progression of disease during treatment
and monitoring a return to normalcy and
FIG. 18G is a summary of the data illustrated in FIGS. 18A-18F.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
Assay Compound
According to the present invention, an assay reagent is
manufactured for determining the activity of an enzyme in a
metabolically active whole cell. The assay reagent must be
compatible with the cell such that the cell will remain
metabolically active for at least the duration of the assay.
The assay reagent comprises at least one assay compound which is
capable of passing through the cell wall. The assay compound must
be small enough that it can be transmitted into the cell. An assay
compound having a molecular weight of less than about 5,000 is
presently preferred.
The assay compound contains a leaving group and an indicator group.
The leaving group is selected for cleavage by the enzyme to be
analyzed. The indicator group is selected for its ability to have a
first state when joined to the leaving group, and a second state
when the leaving group is cleaved from the indicator group by the
enzyme. The indicator group is preferably excitable (caused to
fluoresce) at a wavelength about the visible range, for example, at
wavelength between about 450 to 500 nanometers (nm). The indicator
group will usually emit in the range of about 480 to 620 nm,
preferably 500 to 600 nm and more preferably 500 to 550 nm.
Auto-fluorescence of the cell is most prevalent below about 500
nm.
Indicator groups
The indicator group is preferably derived from fluorogenic and
chemiluminescent compounds. The indicator group should be quenched
when joined to the leaving group. The term quenched means that the
indicator group has almost no fluorescence or chemiluminescence
when joined to the leaving group. When the leaving group is
separated from the indicator group, the resulting indicator
compound will have a fluorescence.
Suitable fluorogenic indicator compounds include xanthine
compounds. Preferably, the indicator compounds are rhodamine 110;
rhodol; and fluorescein. These compounds have the following
structures: ##STR1##
In addition, derivatives of these compounds which have the 4' or 5'
carbon protected are acceptable indicator compounds. Preferred
examples of the derivative compounds include 4'(5')thiofluorescein,
4'(5')-aminofluorescein, 4'(5')-carboxyfluorescein,
4'(5')-chlorofluorescein, 4'(5')-methylfluorescein,
4'(5')-sulfofluorescein, 4'(5')-aminorhodol, 4'(5')-carboxyrhodol,
4'(5')-chlororhodol, 4'(5')-methylrhodol, 4'(5')-sulforhodol;
4'(5')-aminorhodamine 110, 4'(5')-carboxyrhodamine 110,
4'(5')-chlororhodamine 110, 4'(5')-methylrhodamine 110,
4'(5')-sulforhodamine 110 and 4'(5')thiorhodamine 110. "4'(5')"
means that at the 4' or 5' position the hydrogen atom on the carbon
atom is substituted with a specific organic group or groups as
previously listed.
Leaving groups
The leaving group is selected according to the enzyme that is to be
assayed. The leaving group will have utility for assaying many
kinds of cellular enzymes, including proteases, glycosidases,
glucosidases, carbohydrases, phosphodiesterases, phosphatases,
sulfatases, thioesterases, pyrophosphatases, lipases, esterases,
nucleotidases and nucleosidases. For the purposes of this
disclosure the term carbohydrases includes all enzymes which will
hydrolyze a carbohydrate. Enzymes which do not recognize and cleave
a leaving group, such as dehydrogenases and kinases, are not
suitable for assays according to the invention. The enzymes to be
measured can be those which are present in various cell
preparations, enzymes found in cytosols, cell surface enzymes,
cytoplasmic enzymes and cell nucleus (nuclear) enzymes. However, as
will be discussed herein, the assay compounds are particularly
useful for detecting intracellular enzymes in living cells.
The leaving group is selected from amino acids, peptides,
saccharides, sulfates, phosphates, esters, phosphate esters,
nucleotides, polynucleotides, nucleic acids, pyrimidines, purines,
nucleosides, lipids and mixtures thereof. For example, a peptide
and a lipid leaving group can be separately attached to a single
assay compound such as rhodamine 110.
Other leaving groups suitable for the enzyme to be assayed can be
determined empirically or obtained from the literature. See, for
example, Mentlein, R., Staves, R., Rix-Matzen, H. and Tinneberg, H.
R., "Influence of Pregnancy on Dipeptidyl Peptidase IV Activity
(CD26 Leukocyte Differentiation Antigen) of Circulating
Lymphocytes", Eur. J. Clin. Chem. Clin. Biochem., 29, 477-480
(1991); Schon, E., Jahn, S., Kiessig, S., Demuth, H., Neubert, K.,
Barth, A., Von Baehr, R. and Ansorge, S., Eur. J. Immunol., 17,
1821-1826 (1987); Ferrer-Lopez, P., Renesto, P., Prevost, M.,
Gounon, P. and Chignard, M., "Heparin Inhibits Neutrophil-Induced
Platelet Activation Via Cathepsin", J. Lab Clin. Med. 119(3),
231-239 (1992); and Royer, G. and Andrews, J., "Immobilized
Derivatives of Leucine Aminopeptidase and Aminopeptidase M.", The
J. of Biological Chemistry, 248(5), 1807-1812 (1973). These
references are hereby incorporated by reference in their entirety.
Various leaving groups are shown in Table 1.
TABLE 1 - pH.sup.1 SUB. RANGE IONIC FUNCTION CONC. (exper- pH.sup.2
TIME STRG OR ENZYME SUBSTRATES (mM) BUFFER iment) lit COFACTOR
MODULATER INHIBITOR mOSM (MIN) (.mu.) INDICATION CD AminoPept A
(H--L--Asp).sub.2 --Rho 110-2TFA 4.8 Hanks 7.5 7.0 1-5 mM 1 0 mM
280-310 5 .1530 CaCl.sub. 2 CaCl.sub.2 Amistatin I
(H--L--Glu).sub.2 --Rho 110-2TFA Hanks 7.0 280-310 3 AminoPept B
(H--L--Arg).sub.2 --Rho 110-4TFA 3.2 Hanks 8.0 .+-. .2 7.5 NaCl
10.sup.-4 M 280-310 3 137 mM Bestatin (H--L--Cys).sub.2 --Rho
110-2TFA Hanks 7.5 1 mM DTT 280-310 3 AminoPept M (H--L--Ala).sub.2
--Rho 110-2TFA 6.4 Hanks 7.0 7.5 10.sup.-4 M 280-310 5 0.1530
Leukemia CD13 Bestatin (H--L--Ala).sub.2 -4'chloro--Rho 110-2TFA
6.4 Hanks 7.0 7.5 10.sup.-4 M 280-310 5 0.1530 Leukemia CD13
Bestatin H--L--Leu Rhodol--TFA 2.4-3.2 Hanks 5.0-7.0 7.5 1.5 mM
1,10- 280-310 1 0.1494 (H--L--Leu).sub.2 4'chloro-Rho 110-2TFA
2.4-3.2 Hanks 5.0-7.0 7.5 Phenalthroline 280-310 1 0.1494
(H--L--Leu).sub.2 Rho 110-2TFA 2.4-3.2 Hanks 5.0-7.0 7.5 280-310 1
0.1494 CD13 (H--L--Met).sub.2 Rho 110-2TFA Hanks 7.5 56 mM 2,2
280-310 5 CD13 Dipyridyl I (H--Gly).sub.2 Rho 110-2TFA 6 Hanks
7.0-7.5 7.5 1 mM DTE 280-310 5 0.1526 CD13 (H--Gly).sub.2
4'chloro-Rho 110-2TFA 6 Hanks 7.0-7.5 7.5 1 mM DTE 280-310 5 0.1526
CD13 AminoPept N (H--L--Pro).sub.2 --Rho 110-2TFA 6 Hanks 7.5 1 mM
DTE 280-310 5 0.1526 CD13 (H--L--Lys).sub.2 Rho 110-4TFA 2.4 Hanks
7.5 .+-. .2 7.5 280-310 5 0.1490 CD13 (H--L--Lys).sub.2 Rho
110-4TFA 2.4 Hanks 5.5 .+-. .2 7.5 280-310 5 0.1490 (H Gly).sub.2
Rho 110-2TFA 6.4 Hanks 5.5-6.0 7.5 280-310 5 (H--L--Ser).sub.2 Rho
110-2TFA 2.4 Hanks 5.0-6.5 7.5 280-310 5 0.1506 Neg Pro
(H--L--Pro).sub.2 Rho 110-2TFA 6 Hanks 7.5 280-310 5 0.1526 Control
DPP I (H--L--Pro--Arg).sub.2 Rho 110-4TFA 6 MES 5.0-6.5 6.5 ImM DTT
280-310 10 (H--Gly--Arg).sub.2 Rho 110-4TFA MES 5.0-6.5 6.5 ImM DTT
280-310 10 DPP II (H--L--Lys--Ala).sub.2 Rho 110-4TFA 2.0 MES 6.5
.+-. .5 6.5 MgCl.sup.2 Zn DTE Bestatin 280-310 10 0.1019
(H--L--Lys--Ala).sub.2 Rho 2.0 Mes 6.5 .+-. .5 6.5 MgCl.sup.2 Zn
DTE Bestatin 280-310 10 0.1019 110-Sulfo-4TFA
(H--L--Lys--Pro).sub.2 Rho 110-4TFA MES 5.5 6.5 MgCl.sup.2 Zn DTE
Bestatin 280-310 10 (H--L Ala--Pro).sub.2 Rho 110-2TFA MES 5.5 6.5
280-310 10 (H--L Lys--Ala--Lys--Ala).sub.2 Rho 3.2 MES 6.5
MgCl.sup.2 Zn DTE Bestatin 280-310 10 0.1229 110-6TFA DPP IV (H--L
Ala--Pro).sub.2 Rho 110-2TFA 280-310 10 CD26 (H--Gly--Pro).sub.2
Rho-2TFA 2.4 Gly--NaOH 7.5 .+-. .5 8.7 Gly--Pro 280-310 10 0.1449
CD26 1.8 mM (H--L--Lys--Pro).sub.2 Rho 110-4TFA 280-310 10 CD26
(H--L--Ala--Ala).sub.2 Rho 110-2TFA 4.0 Gly--NaOH 8.7 1 mM DTT 3.6
mM 280-310 10 0.1449 CD26 Ala--Ala (H--L--Ala--Ala).sub.2 Rho
110-2TFA 8.7 .+-. .2 8.7 1 mM DTE 280-310 10 (Z--Ala--Ala).sub.2
Rho 110 Gly--NaOH 7.0-8.5 8.0 1 mM DTE 280-310 10 (H--L
Ala--Ala--Ala--Ala).sub.2 Rho Gly--NaOH 8.7 1 mM DTE 280-310 10
CD26 110-2TFA TriPeptidyl- (H--L--Ala--Ala--Arg).sub.2 Rho 110-4TFA
Gly--NaOH 8.7 280-310 10 Pept Cathepsin B (H--L--Gln--Ser).sub.2
Rho 110-2TFA 3.6 MES 6.5-7.5 6.5 1 mM DTE 13 mm-22 mM 280-310 10
0.1055 Breast Leupeptin I Cancer (H--L--Gln--Ser).sub.2 Rho
110-2TFA MES 5.5 .+-. .2 5.5 280-310 10 (H--L--Val--Ser).sub.2 Rho
110-2TFA 4.0 MES 6.5-7.5 6.5 13 mM-22 mM 280-310 10 0.1059 Tumor
Leupeptin I Growth Cystatin C (H--L--Leu--Gly).sub.2 Rho
110-2Tartrate 1.6-2.0 MES 6.0-6.5 6.5 13 mM-22 mM 280-310 10 0.1037
Progression Leupeptin I Cysteine 2 mM (H--L--Leu--Gly).sub.2 Rho
110-2Acetate MES 5.0-6.0 5.5 280-310 10 (H--L--Val--Lys).sub.2 Rho
110-4TFA 1.6 MES 6.5 1 mM DTE 13 mM-22 mM 280-310 10 0.1072 Lung
Tumor Leupeptin I (H--L Leu--Leu--Arg).sub.2 Rho 110-4TFA 6.4 MES
6.5 13 mM-22 mM 280-310 10 0.1232 Leukemia Leupeptin I
(H--L--Leu--Gly--Leu--Gly).sub.2 Rho 6.4 MES 6.5 13 mM-22 mM
280-310 10 0.1083 110-2TFA Leupeptin I
(H--L--Val--Lys--Val--Lys).sub.2 Rho MES 6.5 1 mM DTE Leupeptin I
280-310 10 110-6TFA 1 mM EDTA (H--L--Ala--Arg--Arg).sub.2 Rho
110-6TFA 280-310 10 (H--L--Arg--Arg).sub.2 Rho 110-6TFA 13 mM-22 mM
280-310 10 Gastric Leupeptin Cancer Cathepsin B1
(H--L--Leu--Leu--Arg).sub.2 Rho 110-4TFA 6.4 MES 7.0 .+-. .5 6.5 13
mM-22 mM 280-310 10 0.1232 Smokers Leupeptin
(H--L--Ala--Arg--Arg).sub.2 Rho 110-6TFA MES 6.5 13 mM-22 mM
280-310 10 Leupeptin Cathepsin C (Z--Ala--Gly).sub.2 Rho 110 Gly
7.5 .+-. .5 7.5 280-310 10 (H--L--Ala--Gly).sub.2 Rho 110-2Acetate
2.4 Gly 8.0-8.5 8.7 280-310 10 0.1473 (H--L--Thr--Pro).sub.2 Rho
110-2TFA 2.4 Gly 7.5-9.0 8.7 280-310 10 0.1473 (Z--Thr--Pro).sub.2
Rho 110 6.0-9.0 7.5 280-310 10 (H--L--Pro--Arg).sub.2 Rho 110-4TFA
Gly 8.7 280-310 10 Cathepsin D (H Gly--Leu).sub.2 Rho 110-2TFA 1.2
MES 5.0 .+-. .5 6.5 10 mm 280-310 10 0.1031 Breast Pepstatin II
Cancer (H--L--Thr--Pro).sub.2 Rho 110-2TFA 2.4 MES 5.0 .+-. .2 6.5
280-310 10 0.1043 MS Liver Disease Neutral Endo (H
Gly--Pro--Leu--Gly--Pro).sub.2 Rho 3.2 MES 7.0 .+-. .5 6.5 280-310
10 0.1051 Leuk (ALL) CD10 Peptidase 110-2TFA (H
Gly--Phe--Gly--Ala).sub.2 Rho 110-2TFA Zinc .DELTA. 280-310 10
(CALLA) (H--L--Arg--Gly--Glu--Ser).sub.2 Rho 280-310 10 110-4TFA
EndoPept I (H--L--Arg).sub.2 Rho 110-4TFA .1M Tris 7.5 280-310 10
Hcl (H--L--Glu--Gly--Arg).sub.2 Rho 110-4TFA 280-310 10 EndoPept II
(H--L--Arg--Arg).sub.2 Rho 110-6TFA PO4 7.0 1 mM DTE 280-310 10
(H--L--Ala--Arg--Arg).sub.2 Rho 110-6TFA 1 mM EDTA 280-310 10
Membrane (H--Gly--Ala--Ala--Ala).sub.2 Rho 280-310 10 Assoc.
110-2TFA EndoPept I Membrane (H--L--Arg--Arg).sub.2 Rho 110-6TFA
280-310 10 Assoc (H--L--Ala--Arg--Arg).sub.2 Rho 110-6TFA 280-310
10 EndoPept II Glutathione (H--L--Glu--Cys--Gly).sub.2 Rho 110-2TFA
280-310 10 Chymotrypsin (H--L--Glu--Gly--Phe).sub.2 Rho 110-2TFA
.1M 7.0 PMSF 280-310 10 Tris Trypsin (H--L--Arg).sub.2 Rho 110-4TFA
7.0 Antipain 280-310 10 (H Gly--Gly--Arg).sub.2 Rho 110-4TFA 13
mM-22 mM 280-310 10 Leupeptin Ester (N-Acetyl MET).sub.2 Rho 110
.1M 6.5 280-310 10 Proteinase PO4 .gamma. GT (.gamma.-Glu).sub.2
Rho 110-2TFA 7.0 1 mM 280-310 10 Gly--Gly Elastase
(H--L--Ala--Ala--Tyr).sub.2 Rho .alpha. 1-Antitrypsin 280-310 10
110-2TFA (H--L--Ala--Ala--Pro--Ala).sub.2 Rho 280-310 10 110-2TFA
(H--L--Ala--Ala--Ala).sub.2 Rho 110-2TFA 280-310 10
(H--L--Ala--Pro--Ala).sub.2 Rho 110-2TFA 280-310 10 Plasmin
(H--L--Ala--Phe--Lys).sub.2 Rho 110-4TFA 13 mM-22 mM 280-310 10
Leupeptin I (H--L--Glu--Lys--Lys).sub.2 Rho 110-6TFA 280-310 10
(H--L--Val--Leu--Lys).sub.2 Rho 110-4TFA 280-310 10 Urokinase
(H--Gly--Gly--Arg).sub.2 Rho 110-4TFA 280-310 10
(H--Gly--Arg).sub.2 Rho 110-4TFA 280-310 10 HIV Protease
(H--L--Lys--Ala--Arg--Val).sub.2 Rho 280-310 10 110-6TFA
(H--L--Lys--Ala--Arg--Val--Phe).sub.2 Rho 280-310 10 110-6TFA
v-Thrompsin (H--L--Val--Pro--Arg).sub.2 Rho 110-4TFA PMSF 280-310
10 Pancreatic (H--L--Pro--Phe--Arg).sub.2 Rho 110-4TFA 280-310 10
Cathepsin L (H--L--Phe--Arg).sub.2 Rho 110-4TFA 13 mM-22 mM 280-310
10 Breast Leupeptin Carcinoma growth cancer Cathepsin H
(H--L--Arg).sub.2 Rho 110-4TFA 8.0 .+-. .2 280-310 10 Breast
Carcinoma Collagenase (H--Gly--Pro--Leu--Gly--Pro).sub.2 Rho MES
5.5 .+-. .2 5.5 MgCl.sup.2 DTE Bestatin 280-310 10 110-2TFA Neutral
FL(palmitate).sub.2 .1 Hanks 7.5 280-310 10 .15 Esterase Acidic
Esterase .1 Mes 6.5 280-310 10 .10 Acid FL(phosphate).sub.2
-2NH.sub.4.sup.+ .2 Mes 5.0 280-310 5 .10 Phosphatase Alkaline
FL(phosphate).sub.2 -2NH.sub.4.sup.+ .2 Gly 8.7 280-310 5
Phosphatase Tartrate FL(phosphate).sub.2 -2NH.sub.4.sup.+ .2
Tartrate 5.2 280-310 5 .10 Hairy Cell Resistant Mes Leukemia
Phosphatase Acid Rho 110(phosphate).sub.2 .1 Mes 5.0 280-310 5
Phosphatase Alkaline Rho 110(phosphate).sub.2 .1 Gly 8.7 280-310 5
Phosphatase Tartrate Rho 110(phosphate).sub.2 .1 Tartrate 5.2
280-310 5 Resistant Mes Phosphatase Neutral Non-
Fluorescein(acetate).sub.2 .1 Hanks 7.5 280-310 3 .15 Monocytes,
specific Megakaryo- Esterase cytes, Lympho- Acidic Non- .1 Mes
4.0-6.5 280-310 3 .10 cytes specific Esterase Neutral
FL(propionate).sub.2 .1 Hanks 7.5 280-310 3 .15 Esterase Acid
Esterase .1 Mes 6.5 280-310 3 .10 Neutral FL(chloroacetate).sub.2
.1 Hanks 7.5 280-310 3 .15 Immature Chloroacetate Neutrophils &
Esterase Mast cells Acidic .1 Mes 6.5 280-310 3 .15 Chloroacetic
Esterase Neutral FL(butyrate).sub.2 .1 Hanks 7.5 280-310 3 .15
Monocytes & Esterase I Megakaryo- Acidic cytes Esterase I .1
Mes 6.5 280-310 3 .10 Neutral FL(chlorobutyrate).sub.2 .1 Hanks 7.5
280-310 3 .15 Esterase I Acidic .1 Mes 6.5 280-310 3 .10 Esterase I
Neutral FL(valerate).sub.2 .1 Hanks 7.5 280-310 3 .15 Esterase
Acidic .1 Mes 6.5 280-310 3 .10 Esterase Neutral
FL(hexanoate).sub.2 .1 Hanks 7.5 280-310 3 .15 Esterase Acidic .1
Mes 6.5 280-310 3 .10 Esterase Neutral FL(heptanoate).sub.2 .1
Hanks 7.5 280-310 3 .15 Esterase
Acidic .1 Mes 6.5 280-310 3 .10 Esterase Glycopyrano-
(Acetyl-.alpha.-D-glucopyranosyl).sub.2 Rho 110 .24 Mes 6.8 280-310
10 sidase Glucuronidase (B-D-glucuronide).sub.2 Rho 110 .24 Mes 5.0
280-310 10 Leukemia Galactopyrano- (B-D-Galactopyranoside).sub.2
Rho 110 .24 Hanks 7.5 280-310 10 sidase Tyrosine (H--L-Tyrosine
Phosphate).sub.2 Rho .1 Hanks 6.5 280-310 5 Cell Cycle Phosphtase
110-2TFA Cell Division Serine (H--L-Serine Phosphate).sub.2 Rho .1
Hanks 6.5 280-310 5 Cell Cycle Phosphatase 110-2TFA Cell Division
Threonine (H--L-Threonine Phosphate).sub.2 Rho .1 Hanks 6.5 280-310
5 Cell Cycle Phosphatase 110-2TFA Cell Dividion Neutral
(N-Acetyl-L--Ala).sub.2 FL .1 Hanks 7.5 280-310 5 Monocytic
Esterase II .1 Mes 6.5 280-310 Leukemias 45 RD Adenosine
(Adenosine) Rho 110-2TFA .1 Mes 6.0 280-310 10 AIDS Deaminase
Thymidine (Thymidine).sub.2 Rho 110 .1 Mes 6.0 280-310 10 Deaminase
Cytosine (Cytosine).sub.2 Rho 110-2TFA .1 Mes 6.0 280-310 10
Deaminase Guanine (Guanine).sub.2 Rho 110-2TFA .1 Mes 6.0 280-310
10 Deaminase 5'Nucleotidase (Adenosine Menophosphate).sub.2 Rho .1
Hanks 7.5-9.0 280-310 10 Pap Smear 110-2TFA Adenine Mono- Rho
110(AMP).sub.2 -4NH.sub.4.sup.+ .1 Mes 6.0 280-310 10 AIDS
phosphate Deaminase Angiotensin (Hippuyrl-His--Leu).sub.2 Rho 110
.1 HEPES 8.0 280-310 10 CALLA Converting Enzyme Cholinesterase
FL(Choline).sub.2 .1 Hanks 8.0 Zn.sup.++ 280-310 10 Cholinesterase
FL(Buytryl-Thiocholine).sub.2 .1 Hanks 8.0 Zn.sup.++ 280-310 10
Acetyl FL(Acetyl-Choline).sub.2 .1 Hanks 8.0 Zn.sup.++ 280-310 10
Cholinesterase Nucleosidase (Adenine).sub.2 Rho 110-2TFA .1 Hanks
7.4 280-310 10 Pap Smear Lipase (Saturated Hydrocarbon).sub.2 Rho
110 .1 Lipase (Unsaturated Hydrocarton).sub.2 Rho 110 .1 Lipase
(Triacetin).sub.2 Rho 110 .1 Hanks 7.7 Phospholipase Rho 110
(Phosphatidylcholine).sub.2 -2TFA .1 Hanks 7.0 280-310 10 .15
Phospholipase Rho 110 (Phosphatidylinositol).sub.2 .1 Hanks 7.0
280-310 10 .15 C Phospholipase (Phosphatidyl-choline).sub.2 Rho
110-2TFA .1 Hanks 7.0 280-310 10 .15 D Phospholipase
(Phosphatidyl-choline).sub.2 Rho 110-2TFA .1 Hanks 7.0 280-310 10
.. A (H--L-thyroxine).sub.2 Rho 110-2TFA .sup.1 Range determined
experimentally with cells .sup.2 pH from scientific literature
using cytosol
Preferred peptide leaving groups that react with cellular enzymes
are included in Table 1. As examples, the enzymes
glutamyltranspeptidase reacts with gamma-glutamyl amino acid
peptide giving gamma glutamic acid; trypsin cleaves the peptide at
the arginine residue; aminopeptidase-M hydrolyzes the peptide at
the aliphatic amino acid residue; and chymotrypsin cleaves the
peptide at the phenylalanine residue.
It has been discovered that when the leaving group is a salt
complex, it will significantly improve the transmission of the
assay compound into the cell. The selection of an appropriate salt
complex requires a consideration of the compatibility with the
cell, solubility in the aqueous media, and cleavage by the enzyme.
Particular care is required in the selection of the peptide salt
since even isoenzymes have been found to be specific in their
recognition of particular salts.
Leaving groups for saccharidases are preferably prepared by the
synthesis of monosaccharides, oligosaccharides or polysaccharides
comprising between one and about ten sugar residues of the
D-configuration. Examples of useful sugars are
monosaccharides-pentoses; ribose; deoxyribose; hexose: glucose,
dextrose, galactose; oligosaccharides-sucrose, lactose, maltose and
polysaccharides like glycogen and starch.
The sugar can be an alpha or beta configuration containing from 3
to 7 and preferably 5 to 6 carbon atoms. Analogs of these sugars
can also be suitable for the invention. Preferably, the
D-configuration of the monosaccharide or disaccharide is utilized.
The monosaccharide or disaccharide can be natural or synthetic in
origin.
Leaving groups for nucleases, nucleotidases, and nucleosidases are
preferably prepared by the synthesis of nucleic acids, purines,
pyrimidines, pentoses sugars (i.e., ribose and deoxyribose) and
phosphate ester. Examples are adenine, guanine, cytosine, uracil
and thymine. Leaving groups for restriction enzymes would include
polynucleotides.
The nucleic acids contain a purine or pyrimidine attached to a
pentoses sugar at the 1-carbon to N-9 purine or N-1 pyrimidine. A
phosphate ester is attached to the pentose sugar at the 5'
position. Analogs of these building blocks can also be used.
Leaving groups for lipases are preferably prepared by the synthesis
of simple lipids, compound lipids or derived lipids. Simple lipids
can be esters of fatty acids, triglycerides, cholesterol esters and
vitamin A and D esters. Compound lipids can be phospholipids,
glycolipids (cerebrosides), sulfolipids, lipoproteins and
lipopolysaccharides. Derived lipids can be saturated and
unsaturated fatty acids and mono or diglycerides. Analogs of these
lipids can also be used.
Examples of lipids are: triglycerides--triolein, fatty
acids--linoleic, linolenic and arachidonic; sterols--testosterone,
progesterone, cholesterol; phospholipids--phosphatidic acid,
lecithin, cephalin (phosphatidyl ethanolamine) sphingomyleins;
glycolipids --cerebosides, gangliosides.
Leaving groups for esterases are preferably prepared by the
synthesis of carboxylic acids comprising between 2 and 30 carbon
atoms. The carboxylic acids can be saturated or unsaturated. The
carboxylic acid preferably contains 2 to 24 carbons and more
preferably 4 to 24 carbon atoms. Analogs of theses carboxylic acids
can also be used. The carboxylic acids can be natural or synthetic
in origin. Examples are butyric, caproic, palmitic, stearic, oleic,
linoleic and linolenic.
Leaving groups for phosphatases are preferably prepared by the
synthesis of phosphates, phosphatidic acids, phospholipids and
phosphoproteins. Analogs of these compounds can also be used.
Examples are ATP, ADP, AMP and cyclic AMP (c-AMP).
Leaving groups for peptidases are preferably prepared by the
synthesis of peptides comprising between one and about ten amino
acid residues of the L-configuration. Typically, it has been found
that the synthesis of peptides having more than about six amino
acids produces a low yield. However, where the yield is acceptable,
peptides of greater length can be employed.
The amino acids preferably contain 2-10 and preferably 2-8 carbon
atoms. Analogs of these amino acids can also be suitable for the
invention. If the amino acids are chiral compounds, then they can
be present in the D- or L- form or also as a racemate. Preferably,
the L- configuration of the amino acid is utilized. The amino acids
of the oligopeptide can be natural and/or of synthetic origin.
Amino acids of natural origin, such as occur in proteins and
peptide antibiotics, are preferred. Synthetic amino acids can also
be used, such as pipecolic acid, cyclohexylalanine, phenylglycine,
.alpha.-aminocyclohexylcarboxylic acid, hexahydrotyrosine,
norleucine, or ethionine.
Protecting (Blocking) Groups
Protecting groups are preferably employed when synthesizing the
leaving group to prevent undesired side reactions of the leaving
group during synthesis of the assay compound. N-terminal protecting
groups and polar organic protecting groups on the other portion of
the amino acid molecule are used to prevent undesired side
reactions of the amino acids during syntheses of the peptides. The
protecting groups, also known as blocking groups, are removed prior
to the assay, unless the presence of a particular blocking group or
groups is found not to interfere with the assay.
The N-terminal protecting groups include an arylcarbonyl,
alkylcarbonyl, alkoxycarbonyl, aryloxycarbonyl, aralkoxycarbonyl,
arylsulfonyl, alkylsulfonyl, or other equivalents known to those
skilled in the art of peptide syntheses. The polar organic
protective groups include hydroxyl, guanidinyl, sulfhydryl and
carboxyl or other equivalents known to those skilled in the art of
peptide syntheses. Gross and Meienhofer, eds., The Peptide, 3(3-81)
(Academic Press, New York, 1981), describe numerous suitable amine
protecting groups.
Preferred examples of the N-terminal blocking groups include
formyl, acetyl, trifluoroacetyl, benzyloxycarbonyl, phthaloyl,
benzoyl, acetoacetyl, chloroacetyl, phenoxycarbonyl, carbobenzoxy,
substituted benzyloxycarbonyl, tertiarybutyloxycarbonyl,
isopropyloxycarbonyl, allyloxycarbonyl, phthaloyl, benzoyl,
acetoacetyl, chloroacetyl, phenoxycarbonyl, methoxysuccinyl,
succinyl, 2,4-dinitrophenol, dansyl, p-methoxybenzenesulfonyl, and
phenylthio.
Preparation of Intermediate Complex
A compound containing a blocking group and a leaving group such as
an amino acid is reacted with an agent to form an active
intermediate complex. The leaving group is selected based on the
leaving group desired in the final assay compound. Suitable agents
are known to those skilled in the art of peptide chemistry.
Examples of suitable agents include carbodiimides, (preferably
1-ethyl-3-(3'-dimethylaminopropylcarbodiimide hydrochloride) and
benzotriazolyl-N-oxy-tris(dimethylamino)phosphonium
hexafluorophosphate (BOP reagent) and 1-hydroxybenzotriazole (HOBT
reagent). The reagents are typically stirred in a flask at room
temperature. The chemical structure of the intermediate complex is
presently unknown. The presence of the complex can be confirmed by
thin layer chromatography.
Preparation of Reaction Product
The intermediate complex is further reacted with a compound
containing an indicator group (indicator compound) to form a
reaction product. As appreciated by those skilled in the art of
peptide chemistry, the indicator compound is dissolved in a solvent
to facilitate the reaction with the intermediate complex. The
reagents are typically stirred in a flask at room temperature for a
time sufficient to form a reaction product. The reaction product
can be confirmed by developing a thin layer chromatography (TLC)
plate in an organic solvent. The reaction product should be a
non-fluorescent compound. When the indicator group is rhodamine
110, rhodol or a derivative, the presence of the reaction product
is confirmed by contacting the reaction product with an acidic
solution, such as hydrochloric acid, which cleaves the leaving
group thereby forming a colored product. When the indicator group
is fluorescein or a derivative, the presence of the reaction
product is confirmed by contacting the reaction product with a
basic solution, such as sodium hydroxide, which cleaves the leaving
group thereby forming a colored product. If only one spot on the
TLC plate gives a positive test and there are no trace amounts of
fluorescence or its derivatives, the reaction product is of
acceptable purity for this stage of the process.
Purification of Reaction Product
The reaction product is then separated from other side reaction
products, by-products and starting materials in the following
manner. Preferably, the reaction product is concentrated to an oil
under reduced pressure so as to remove volatile solvents that might
be present. The reaction product oil is then redissolved in a
minimum of an organic solvent, preferably chloroform, methylene
chloride, and further separated from the other side reaction
products, by-products and starting materials by normal phase
preparative high pressure liquid chromatography (HPLC). Other
conventional methods of separation can be employed. Separation of
the reaction product is verified by TLC, as previously described,
and analytical reverse phase HPLC. The reverse phase HPLC will
depict the presence of one major band of reaction product.
The reaction product is separated from the other side reaction
products, by-products and starting materials so that the reaction
product can be further processed by having the blocking groups
removed. If the reaction product is not sufficiently separated from
the other side reaction products, by-products and starting
materials, then a low yield of the assay compound containing an
indicator group and leaving group will be obtained. Moreover, the
quality of the separation will have an effect on the amount of
purification that will be subsequently necessary to obtain an assay
compound for use in the metabolically active cell.
Removal of Blocking Group
The blocking group which is blocking (protecting) the leaving group
is then removed from the reaction product to obtain an assay
compound ("intermediate compound" if a salt is to be formed) which
contains an indicator group and a leaving group. The reactions are
conducted to obtain a free amino acid xanthine derivative by
methods known to those skilled in the art. When the blocking group
on the indicator group comprises benzyloxycarbonyl (CBZ), the
blocking group is removed by a catalytic reaction of the reaction
product in an organic solvent with hydrogen in the presence of
palladium or platinum. Further details of this process are shown in
Example 13. When the blocking group on the indicator group
comprises 9-fluorenylmethyloxycarbonyl (FMOC), the blocking group
is typically removed by the reaction of the reaction product in a
polar solvent with an organic base. Further details of this process
are shown in Example 1.
To confirm that the blocking group has been removed from the
resulting intermediate compound, the intermediate compound is
analyzed by analytical reverse phase HPLC. In addition, the
resulting intermediate compound can be further confirmed by
developing a thin layer chromatography plate in an organic
solvent.
Physiologically Acceptable Salt Formation
This intermediate compound having an indicator group and leaving
group is then reacted with an acid or a base to form an assay
compound, which is a physiologically acceptable salt. It is
important according to the method of the invention that the assay
compounds be physiologically acceptable to the cell. The selection
of the acid or base has a material affect on whether the resulting
assay compound will be physiologically acceptable to the cell. In
addition, it has been found that the selection of the acid affects
the selectivity of the assay compound for the enzyme to be
assayed.
It has been found that hydrogen bromide (HBr), even when buffered,
kills cells. To confirm whether an acid will be appropriate to use,
a selected acid is used to make an assay compound. The assay
compound is then tested with a metabolically active cell to
determine if viability (Trypan Blue; propidium iodide-fluorescein
diacetate [PI-FDA]) over the assay time period is affected.
Viability is confirmed with Trypan Blue or PI-FDA over a time
period of 10 seconds to 30 minutes. If the viability of the cell
sample at between one and three million cells/mL decreases by10%
then the salt of the compound is rejected and another salt of the
assay compound is synthesized.
Preferably the acid that is used to form the salt is selected from
the group consisting of hydrochloric, sulfuric, nitric, maleic,
acetic, trifluoroacetic, tartaric acid, citric, succinic and
p-toluenesulfonic acid. More preferably the acid is selected from
the group consisting of acetic, trifluoroacetic, tartaric acid, and
p-toluenesulfonic acid. Most preferably the acid is
trifluoroacetic. When a base is used, ammonia or organic bases can
be used. Most preferably, the base is ammonia.
Purification of the Assay Compound
The assay compound is purified, preferably by reverse phase HPLC.
It is very important that the side reaction products, by-products
and starting materials from the synthesis of the assay compound be
removed which would diminish the utility of the assay.
Non-physiologically acceptable impurities should be removed. In
addition, the background noise generated from impurities should be
less than the auto-fluorescence of a metabolically active cell.
It has been found that when a leaving group is present as an
impurity, the leaving group can be an inhibitor to enzyme activity.
Still further, metal impurities in any of the starting materials
can poison the enzymes, prevent hydrolysis of the assay compound
and interfere with the accuracy of the enzyme assay.
In addition, impurities will create background fluorescence which
will add to the natural fluorescence of the cell to create a level
of background noise which can interfere with the detection of
enzyme generated fluorescence. Fluorescent impurities can be taken
up by the cell, and a rate measurement of fluorescence against time
will show a false rate of increasing fluorescence that is due only
to this cellular uptake of fluorescent impurities. This is a
particular problem if the assay is conducted to determine the
presence or absence of an enzyme, since this impurity will indicate
a rate of fluorescence which will falsely appear to be attributable
to enzymatic activity.
The assay compound can be purified by techniques known in the art.
As shown in Example 1, the purification of rhodamine 110 substrate
can be accomplished by reverse phase column chromatography.
In the case of the preparation of salts of peptide-rhodamine 110
compounds, a significant level of impurities is created. These
impurities include free indicator compound, monosubstituted
rhodamine 110, blocked amino acids and peptides.
The fluorescence impurities should be removed to a level that they
do not obscure the baseline detection of the enzyme in the cell.
The baseline detection can be established by analyzing log
dilutions of an indicator group. Preferably the impurities should
be removed so that the fluorescence of the impurities is less than
the auto-fluorescence of the metabolically active cell.
Assays for peptidases using assay compounds generate fluorescence
generally in the range of 10.sup.-5 to 10.sup.-6 Molar free
rhodamine 110. Therefore, it is preferred that the free rhodamine
110 and blocked peptide impurities in the assay reagent should be
removed to a concentration of less than the fluorescence generated
by about 1.times.10.sup.-6 M and more preferably less than the
fluorescence generated by about 10.sup.-7 Molar free indicator
group. This amounts to a 100,000 photon count using rhodamine 110
as a standard at 10.sup.-7 -10.sup.-8 M, preferably
5.times.10.sup.-8 M in a 1 cm path length cuvette when measured
over 10 min. on a photon counting spectrofluorometer manufactured
by the SLM Company of Chicago, Ill. This corresponds to a use level
on the flow cytometer where no cellular false positive can be
detected for a 10 minute period at the highest sensitivity setting.
In the case of the peptide-rhodamine 110 compounds, this has been
found to require a concentration of impurities of less than one
part per one hundred thousand, more preferably less than one part
per five hundred thousand, most preferably less than one part per
million. The presence of impurities causes a decrease in the
storage stability of the compound, resulting in an increased
autohydrolysis which leads to increased background fluorescence. A
compound should be free of impurities such that when the compound
(or reagent containing the compound) is stored at 4.degree. C. for
30 days, preferably 90 days, more preferably 180 days, most
preferably one year, the background fluorescence increases less
than 10%, preferably less than 5%, most preferably less than 1%
over these time periods, respectively. The purified compound or
lyophilized reagent are stored in a sealed container over dry
nitrogen under atmospheric pressure. The starting point in time for
measuring stability is usually immediately after purification of
the assay compound is completed but it can be any time such as
immediately after the preparation of the assay reagent is
completed.
Normal phase preparative HPLC procedures are presently preferred to
separate peptide-indicator compound from the impurities. As is
known in the art, solvents of varying polarity can be mixed in
varying concentrations in order to more effectively separate the
peptide-indicator compound from the various impurities. Thin layer
chromatography (TLC) can be utilized to test for the presence of
the rhodamine 110 substrate in the eluate. This is done by placing
a drop of the eluent on the TLC plate, and then treating the spot
with a suitable acid, such as HCl, to detect the presence of the
rhodamine 110 substrate, which will turn bright yellow when treated
with acid. Analytical reverse phase high pressure liquid
chromatography is used to test the peptide-indicator product for
purity, as evidenced by a single sharp band in the absorption
spectrum.
Preparation of Assay Reagent
The assay reagent must be compatible with the metabolically active
cell. The assay reagent should have an osmolality of from about 250
milliosmoles to 350 milliosmoles, preferably from about 275
milliosmoles to 320 milliosmoles. Further, the assay reagent will
have an ionic strength between about 0.1 to 0.3 .mu.. In addition,
the pH of the assay reagent will be between about 4.0 and 9.5,
preferably between about 5.0 and 8.0. The preferred pH for assay
compounds for particular enzymes is included in Table 1.
It has been further found that the efficacy of an intracellular
assay is substantially improved by the addition of one or more
components in the assay reagent. Examples of improvements include a
reduction of reaction time, increased selectivity for the targeted
enzyme, reduction of competing enzyme reactions, increasing signal
of enzyme reaction, increasing reactivity of the assayed enzyme
relative to other non-targeted enzymes, increasing the retention
time of the indicator group within the cell and other similar
advantageous results.
Additional components include buffers, cofactors, modulators,
inhibitors, activators for increasing activity of the target
enzymes over other non-targeted enzymes, solubilizing components
and retention components can be included in the assay reagent to
improve the enzyme assay results. These components are
physiologically acceptable to the metabolically active whole cell
that is being assayed.
The chemical nature of the buffer is important to the reactivity of
the assay compound with the cellular enzymes. For example, it has
been found that Hanks solution is a better cellular buffer than
cacodylic acid at 0.1M concentration for amino peptidase. More
specifically, by utilizing Hanks solution, at pH 7.5, it has been
further found that the assay compound has a higher sensitivity for
the targeted enzyme. In addition, the assay compound hydrolysis by
the enzyme occurs at an increased rate of reaction. Although Hanks
solution contains calcium chloride at a concentration of 1.26 mM,
calcium chloride has been found in the case of aminopeptidase to be
inhibitory to the enzyme reaction with the assay compound,
(H-L-Asp).sub.2 rhodamine 110, at concentrations of approximately
10 mM.
Buffer components that show no inhibitory effect to the cells can
be used. Suitable buffer components are
N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid (TES), Hanks
balanced salt and 2-N-morpholinoethanesulfonic acid (MES),
tris-glycine, HEPES, glycine sodium hydroxide, and cacodylate. The
preferred buffer components are MES for acidic solutions, Hanks for
neutral solutions, and glycine sodium hydroxide for basic
solutions. Preferred buffers for particular enzymes are included in
Table 1. A metabolic energy source such as a sugar (glucose) can be
added.
Cofactors are components not consumed in the enzymatic reactions,
but are required to make the enzyme function. Suitable cofactors
include metals such as Ca.sup.+2, Zn.sup.+2, Mg.sup.+2, Fe.sup.+2
and Mn.sup.+2. These cofactors can increase the selectivity of the
enzyme for the leaving group. The cofactors can also be co-enzymes
or vitamins. Preferred cofactors for particular enzymes are
included in Table 1.
Modulators are components used to decrease the sensitivity of the
enzyme for the leaving group. The modulators speed up or slow down
the activity of an enzyme by changing the active site. Therefore,
enzyme activity can be down-regulated, as in negative feedback
inhibition by the leaving (stimulated) group inhibiting the
original enzyme. For example, dithioerythritol (DTE) at 1 mM
decreases the sensitivity of the substituted rhodamine 110
substrates containing the amino acids Pro, Gly, Gln-Ser,
Val-Lys-Val-Lys, Ala-Ala, and Ala-Ala-Ala-Ala, but does not change
the sensitivity of the leaving group for the enzyme where the
substrate contains the amino acids Ala-Gly, and Leu-Gly.
Dithiothreitol (DTT) has also been found to be an effective
modulator. Preferred modulators for particular enzymes are included
in Table 1.
Inhibitors and poisons (or toxins) are components that can be added
to reduce the activity of non-targeted enzymes that provide
competing reactions for the leaving group. Inhibitors are usually
very selective for a particular enzyme. For example, EDTA only
works with enzymes requiring Ca.sup.+2, Mg.sup.+2 and some other
metals. Other examples of an inhibitor are bestatin, which
selectively inhibits aminopeptidase and leupeptin which selectively
inhibits cathepsin B. In addition, the monopeptide reagent contains
approximately 137.9 mM per liter of sodium chloride. However, the
addition of 7mM of sodium chloride to the dipeptide reagent has a
slightly inhibitory effect over the pH range of 6.5 to 8.7.
Preferred inhibitors for particular enzymes are included in Table
1.
The assay compound must be soluble in the aqueous media. Solubility
is measured by light scatter using the percent transmittance of
light (or absorbance) through the mixture of the media and assay
compound. As measured on a spectrophotometer, the assay compound
should have a background color at a concentration to be used in an
assay of less than 1000, preferably less than 800, and most
preferably less than 500 milliabsorbance units at 340 nanometers
(25.degree. C.) blanked against distilled or deionized water. The
assay compound will usually be used at a concentration of 0.5 to 10
mM. A useful concentration for determining solubility is 5mM.
Preferably, a two fold excess quantity of the assay compound that
will react with the enzyme during the time of the assay must be
soluble in the aqueous media. An excess of assay compound is
preferred. If an insufficient amount of the assay compound is
provided, the enzyme reaction will completely hydrolyze the assay
compound and the dynamic range of the assay will be limited. The
resulting indicator compound will have a limited fluorescence
duration. However, when an excess of the assay compound is
employed, the enzyme reaction will continuously hydrotyze the assay
compound and the fluorescence duration will continue during the
enzyme reaction. This provides the advantage of having a longer
time period in which to sense for one or more reaction states of
the assay compound.
A solubilizing agent can be utilized with assay compounds for which
salts are not available, or where such solubilizing agents will
assist the transfer of the assay compound into a metabolically
active cell. The solubilizing component is present in an amount
effective to enable the assay compound to pass through the cell
lipid bilayer without detrimentally affecting the cell. The
solubilizing agent should be carefully chosen because the wrong
solubilizing agent can cause lysis or cell death.
When the assay compound has a background color (at the
concentration to be used in an assay) greater than 1,000, greater
than 800 or greater than 500 milliabsorbance units, a solubility
component may be used to lower the background color to less than
1,000, less than 800 or less than 500 milliabsorbance units.
However, the concentration of the solubilizing component is
limited. If a high concentration of the solubilizing component is
used, metabolically active cells will be lysed. If a low
concentration of the solubilizing component is used, sufficient
solubility of the assay compound will not be attained. The
effective amount of solubilizing component may be empirically
determined, but is typically less than 10.0% by weight of the assay
compound.
Suitable solubilizing components include non-ionic surfactants,
polyethylene glycol, dimethyl sulfoxide (DMSO), and mannitol, as
noted in Table 2. BRIJ 35 and TWEEN 20 are the tradenames for
products from ICI Americas, Inc. PLURONIC 25 R8 is the tradename
for a product from BASF Wyandotte. TRITON X100 is the tradename for
a product from Rohm and Haas Company.
TABLE 2 ______________________________________ COMMERCIAL
CONCENTRATION NAME CHEMICAL STRUCTURE
______________________________________ 0.1% BRIJ 35 Polyoxyethylene
lauryl (nonionic) ether 0.2% PLURONIC Ethylene oxide with 25 R8
hydrophobic base from (non-ionic) propylene oxide and propylene
glycol 0.1% TRITON X100 Octylphenoxy polyethoxy (non-ionic) ethanol
0.1% TWEEN 20 Polyoxyethylene sorbitan (non-ionic) monolaurate
(polysorbate 20) 0.1% Polyethylene Glycol 5% Dimethyl Sulfoxide
4.5% Mannitol ______________________________________
When using a solubilizing component, certain difficulties have been
encountered. While the solubilizing component facilitates the
transmission of the assay compound into the metabolically active
cell, the solubilizing component will also facilitate the expulsion
of the fluorescent indicator group compound from the metabolically
active cell. The expulsion of the indicator group will have the
negative effect of permitting non-enzyme containing cells to absorb
free dye. When this occurs, the accuracy of an enzyme assay is
compromised.
In addition, the electronic configuration and polar nature of the
liberated indictor dye influences its ability to be retained within
the cell. Retention of the dye is important for proper
detection.
A feature of the present invention used to avert the problem of
cellular expulsion when using a solubilizing component, is for the
assay reagent to include a retention component. The retention
component will comprise at least one agent that will inhibit a cell
pump mechanism for expressing extracellular material. Such cell
pumps include the multiple drug response pump, calcium channel
pump, sodium pump, potassium pump and ATPase pump. Suitable
retention components include trifluoperazine.cndot.HCl,
prochlorperazine.cndot.emaleate, and chlorpromazine.cndot.HCl to
inhibit the multiple drug response pump;
verapamil.cndot.hydrochloride to inhibit the calcium channel pump;
and digoxin (C.sub.41 H.sub.64 O.sub.14), digoxin derivatives, such
as ouabain (C.sub.29 H.sub.44 O.sub.12), and strophatidin (C.sub.23
H.sub.32 O.sub.6) to inhibit the sodium, potassium and ATPase
pump.
The media in which the assay compound is dissolved must be
compatible with the cell so that the cell can remain metabolically
active in the media for at least the duration of the assay. The
media is preferably sterile and free of endotoxin and chemicals
that adversely affect the physiology of the cell. The assay
compound is preferably completely soluble in the media at the
concentration at which it is used. The assay compound is preferably
used in concentrations up to the saturation or the suspension level
or before turbidity occurs. The media may be physiological saline
or a buffered solution (phosphate buffered Saline) in which the
assay compound and other additives are dissolved. The media should
preferably include a buffer agent so that the pH of the assay
mixture of metabolically active cells and assay compound is
maintained at a point that is appropriate for the enzyme
hydrolysis.
For storage purposes the compound and media mixture should be
lyophilized under conditions where sublimation of the solvent
occurs upon application of a vacuum. Applying a vacuum to the
sample at a temperature where a liquid forms on the solid before
going to a gas phase, referred to as "melt back" may cause
degradation of the compound. Appropriate temperatures should be
determined for each compound, and preferred temperatures are
usually -5.degree. C. to -35.degree. C. for predominantly aqueous
solutions. During the thermal cycle of lyophilization, heat may be
applied after sublimation to drive off any additional moisture. The
product temperature should never exceed the heat applied and the
product should be brought to room temperature over 15 to 72 hours.
The vacuum should be returned to atmospheric conditions by bleeding
in dry nitrogen. The product is stoppered at atmospheric pressure
and temperature. The lyophilized compound is stored at 4.degree. C.
to 8.degree. C. and may be reconstituted using endotoxin-free
deionized water.
Auto-hydrolysis, which is the nonspecific hydrolysis of the
substrate, yields cellular fluorescence not derived from the target
enzyme. Stability of the substrate compound has been demonstrated
to be a key factor in preventing auto-hydrolysis.
The assay compound and/or the assay reagent should be sufficiently
stable so that no auto-fluorescence or chemiluminescence is created
by the degradation of the assay compound prior to cleavage by the
enzyme. Preferably, when the assay compound or assay reagent is
stored at 20.degree. C. for 30 days, preferably 90 days, more
preferably 180 days and most preferably one year, the reagent
exhibits a photon count of 100,000 or less. Photons can be measured
by using a 2 millimolar solution of assay compound in deionized
water and a path length of 1 cm against a rhodamine 110 standard as
previously described. Fluorescent impurities should account for
less than 10% of the fluorescence generated during the assay.
An acceptable reagent should have the following three
characteristics; (1) there should be a low level of native free
fluorescence that is absorbed by the cells, non-specifically. Thus,
there should be a low level of fluorescent impurities such as free
indicator compounds. The acceptable and preferred levels of these
impurities have already been described. (2) The reagent should be
stable over time so that it does not need to be used shortly after
it is prepared. Certain impurities and certain reagent additives
can increase the rate of autohydrolysis which increases the
fluorescence of the reagent. Acceptable and preferred stabilities
have already been discussed. (3) The reagent should also have a
high enough rate of reaction with the enzyme being measured so that
fluorescence generated as a result of reaction between the enzyme
and the reagent can be easily measured. In one aspect, the reaction
rate should be sufficiently high that fluorescence generated as a
result of cleavage of the leaving group inside the cell is at least
2 times, preferably at least 10 times, more preferably at least 50
times and most preferably at least 100 times greater than other
non-specific fluorescence generated in the assay. In another
aspect, the reagent should contain an unblocked assay compound
which has a reaction rate-which is at least 2 times, preferably at
least 5 times, more preferably at least 100 times, most preferably
at least 1000 times the reaction rate of a corresponding blocked
assay compound. For example, the unblocked assay compounds of the
present invention which contain unblocked amino and or peptide
leaving groups have an enzymatic reaction rate which is
considerably greater than the reaction rate of the corresponding
compound wherein the amine group(s) on the leaving group is blocked
by, for example, a Cbz group.
Types of Assays
It has been discovered that the assay reagent can be used to
determine enzymatic activity of metabolically active whole cells to
provide indication of the presence of a disease, of the progress of
a disease, the efficacy of a drug, and cell differentiation.
It has been found that the activity of one or more enzymes changes
with disease progression. Changes in the activity of one or more
enzymes can be examined to provide an indication of the presence
and progress of a disease. In addition, the measurement of the
activity of certain enzymes can provide an indication of the
response to certain drugs or treatments, since the activity of one
or more enzymes will change if the drug is successfully fighting,
modulating or treating the disease. Still further, it has been
determined that differentiation of a cell can be determined by the
presence of one or more selected enzymes.
Existing tests for the presence of a disease, progress of a
disease, or efficacy of a drug require significant extracellular
concentrations of the enzymes that are being measured. Usually,
hours or days are required to allow extracellular concentrations to
rise to detectable levels. The present invention has the further
advantage of providing a method to produce a reagent for measuring
the intracellular concentrations of enzymes. This enables the
diagnostic assay to obtain analysis of the enzyme of interest in a
shorter period of time and to monitor intracellular events as they
are occurring.
Enzymatic assays are performed by contacting metabolically active
cells with an assay reagent. The leaving group is selected to be
one which can be cleaved from the indicator group by the targeted
enzyme. The reaction occurs for a period of time sufficient for the
leaving group to be cleaved from the indicator group by the
targeted enzyme. Sensing for one or more reaction states confirms
cleavage of the indicator group by the enzyme.
In some instances, one can characterize a disease, the progress of
a disease, or efficacy of a treatment by sensing for only a single
enzyme, as in the case where a disease is characterized by the
presence or lack of activity of an enzyme. For instance, Hereditary
Non-Spherocytic Hemolytic Anemia (HNSHA) is distinguished from
hereditary spherocytosis by the fact that red blood cells are
morphologically normal and manifest a normal osmotic fragility.
Only in the case Of pyrimidine 5'nucleotidase deficiency is the
erythrocyte morphology changed to a basophilic stippling. (See
Example 27) However, in most instances these conditions will be
characterized by assaying for the activity of at least two enzymes.
In practice, five or more enzymes will be used in a panel to serve
as checks and to reduce the probability of false positive or false
negatives since the activity of a targeted enzyme can be present in
two different diseases. However, as the number of targeted enzymes
are increased in the panel, the assay becomes more reliable or
differentiable. The detection is more reliable because two
different diseases will have different enzyme patterns.
Thus, there are at least two ways to run the assay of the present
invention:
running a single assay and detecting a difference between the
beginning state and the end state of a substrate, such as the
cleavage of a single substrate by a target enzyme to yield free
peptide and fluorescent indicator dye; and
running a series of assays with a pattern matrix of several
substrates reacted with an abnormal cell versus the same matrix
reacted with a normal cell.
"Reliability" refers to the ability to make pattern matrix
decisions without failure. Error in a single test may not, in fact,
invalidate a pattern matrix. For a small set of assays, the assay
provides an increased capability to differentiate states of
abnormality.
The panel of selected enzymes are created by developing a range of
normal values for enzymatic activities and ratios of enzymatic
activities to each other. This panel will be used to compare the
test results from the cell analyte. The enzyme activities from the
examined cell analyte is compared to at least one of a
reference/non-diseased cell or a reference/diseased cell to obtain
an indication of a diseased state.
In other instances, the analysis of cellular enzymes involving
classes of enzymes provides the ability to sort cells by type or
morphology. As many as a thousand different enzymes may be
operative in any given cell, but only a few dozen enzymes define
the unique function or functions of any one cell type. Many enzymes
are inhibited or missing from functionally different cells.
Determination of proteases, glycosidases, glucosidases,
carbohydrases, phosphodiesterases, sulfatases, thioesterases,
pyrophosphatases, nucleotidases, nucleosidases, saccharidases,
esterases, phosphatases, lipases and combinations thereof provides
a matrix to rank cells by their functional activity.
Classification of normal cells morphologically can be made by
determining key enzyme activities. For example, nucleated red blood
cells (NRBC's) can be distinguished from non-nucleated red blood
cells (RBC's) by determining dipeptidyl peptidase IV activity in
the cell analyte. In NRBC's dipeptidyl peptidase IV activity will
be present but in RBC's dipeptidyl peptidase IV activity will be
absent. In addition, the age of RBC's can be determined by the
presence or absence of adenosine deaminase or 5'nucleotidase.
In still other instances, the analysis of cellular enzymes
involving classes of enzymes provides the ability to study cell
proliferation. Cell proliferation is stimulated by growth factors.
Cell proliferation is the ability of cells to divide and increase
their numbers. Phases of cell division are under gene control and
take a specified time period for each part of the division process.
The time from one division to the next includes a randomly variable
component. Different cell types require different growth factors in
order to divide. All cells compete for growth factors. Cells are
programmed for a certain number of divisions and then they die.
Abnormal cells that disobey normal constraints on cell division
proliferate to form tumors in the body. They also appear
transformed in cell culture. Cell transformation is often
accompanied by mutation or over-expression of specific
oncogenes.
Some normal cells proliferate as part of their function. Signaling
molecules are produced in the course of the inflammatory response
and stimulate the bone marrow to produce more leukocytes. This
regulation tends to be cell type specific. More specifically, some
bacterial infections cause a selective increase in neutrophils,
while infections from parasites cause a selective increase in
eosinophils.
A blood cell differential can be constructed using this invention
to determine cell types, immature cells, mature cells, abnormal
cells due to drug interreaction and abnormal cells due to disease.
For example, cell types can be identified by the assay of the
present invention. For example, lymphocytes can be distinguished
from monocytes or neutrophils by peroxidase activity. Lymphocytes
will not show peroxidase activity while neutrophils will show
peroxidase activity, and lymphocytes will not show esterase
activity while neutrophils will show esterase activity. In
addition, acetate esterase activity is present in monocytes that
have been inhibited with sodium fluoride, but absent in
neutrophils.
In addition, the analysis of cellular enzymes involving classes of
enzymes provides the ability to study cell activation. Activation
of T cells is a complex process involving various secreted proteins
called interleukins which act as chemical mediators. Activation is
thought to begin when the T cell. stimulates the antigen presenting
cell to secrete one or more interleukins. IL-1 mediator causes the
T cell to stimulate its own proliferation by inducing it to secrete
a growth factor IL-2, as Well as synthesize IL-2 receptor to
initiate proliferation.
Helper T cells are essential for B cell antibody response. Once
activated by foreign antigen, the T cell presents the antigen to a
B cell for antibody synthesis. Other Helper T cells secrete .gamma.
interferon which attracts macrophages and activates them to defend
against infection by microorganisms. Diagnosis of infection from
inflammation and inflammatory diseases has been achieved using the
assay reagent in a pyrogen-free, sterile environment. Activation
and proliferation agents, like phorbol myristate acetate (PMA),
f-Met-Leu-Phe, IL-1, IL-2, GMCSF and .gamma. interferon are added
to the media and specific cell types are observed for response.
Treatment regimes can also be monitored for effectiveness by using
the assay reagent in conjunction with growth stimulators or signal
peptides.
The present invention has potential use in the following clinidal
applications: diagnosis of cervical cancer, diagnosis of viral
replication in HIV patients, diagnosis of HIV infected blood in
blood supply, diagnosis of TB infected HIV patients, improved blood
differential, differential diagnosis of vital from bacterial
infections, differential diagnosis of Lupus from rheumatoid
arthritis, differential diagnosis between rheumatoid arthritis from
osteo arthritis, diagnosis of vasculitis, diagnosis of
cardiovascular disease, monitoring of chemotherapeutic efficacy,
diagnosis of Hodgkins Disease, confirmation of gene implantation
and diagnosis of transplant rejection.
For a diagnosis of cervical cancer, several enzymes related to the
presence of cervical cancer can be measured.
For a diagnosis of viral replication in HIV. patients, HIV
replication in blood cells can be monitored. A sensitive measure of
HIV replication can be important as a predictor of rapid movement
into the AIDS state from the HIV infected stage of the disease.
Since the virus replicates in the lymphocytes and monocytes,
monitoring specific enzyme levels can make the monitoring both
inexpensive and reproducible.
Identification of infected units in blood supply is one of the
major goals of those responsible for the quality of blood supplied
for transfusion to reduce the probability of HIV or Hepatitis
infection. A low cost screening methodology can be devised whereby
the blood can be subjected to HIV antibody testing and testing by
the method of this invention.
In the management of AIDS patients, the early diagnosis of
Tuberculosis is important to insure rapid recovery and to reduce
the chance of further complications. The objective of such a test
using this invention is to distinguish TB.sup.+ HIV patients from
TB.sup.- HIV patients. The early identification of the TB.sup.+ HIV
patients can permit administration of therapy to prevent additional
complications in these immune deficient patients.
This invention also has utility for the differential diagnosis of
viral from bacterial infections. Many patients have an elevated
temperature and it is not known whether the temperature is from a
viral or bacterial origin. The differential diagnosis between viral
and bacterial infections assists the clinician in the management of
these patients by allowing the physician to apply the proper
therapy on an as needed basis.
This invention has further utility for differential diagnosis of
Lupus from rheumatoid arthritis/drug monitoring in rheumatoid
arthritis and Lupus patients. In the early course of disease, the
symptoms for Lupus Erythematosis and rheumatoid arthritis are
sufficiently similar that differential diagnosis of the disease is
difficult, especially when a Lupus patient has early arthritic
involvement. This has clinical consequences since it delays the
administration of the correct therapy. Lupus can be a clinically
aggressive disease and it is beneficial to the patient to have the
correct diagnosis at an early date. These patients have different
enzymes in activated states meaning that this methodology is the
modality to use for a differential diagnosis. Additionally,
monitoring the therapeutic application of steroid drugs can be of
benefit to the patient.
This invention has still further utility for differential diagnosis
between rheumatoid arthritis from osteo arthritis. Rheumatoid
arthritis is an aggressive autoimmune disease which results in
destruction of the panus of the joint. Osteo arthritis is a
degenerative disease of the aging joint which is not immune
mediated. Since immune cells migrate throughout the body, this
methodology provides an early differential diagnosis between these
two diseases. This is important since the correct therapy for each
disease is different.
Moreover, this invention has utility for diagnosis of vasculitis.
Vasculitis is an autoimmune disease of blood vessels generally in
the extremities. Patients with this disease typically have
nondescript complaints of pain which do not permit diagnosis until
considerable damage has been completed on the vascular system by
the immune cells. Since it is an autoimmune disease caused by
circulating immune cells, the disclosed methodology can provide the
needed information to make an early diagnosis.
Furthermore, this invention has utility for monitoring of
cardiovascular disease. Atherosclerosis results in the deposition
of platelets and other cellular components into the walls of
coronary vessels. This process results in the loss of elasticity of
the vessels and eventually in death. It has been shown that in
these patients, as many as 20% of the platelets are in the
activated state. Evaluation of platelets can permit the
identification of patients with active atherosclerotic processes
ongoing and permit administration of disease altering drugs.
Moreover, this invention has utility for monitoring of
chemotherapeutic efficacy. Patients undergoing chemotherapeutic
therapy have altered enzyme patterns which indicates that this
change in enzyme levels can be used to monitor the effectiveness of
chemotherapy.
In addition, this invention has utility for diagnosis of Hodgkins
disease. The practice of this invention can be useful to monitor
the stages of Hodgkins disease.
Furthermore, this invention has utility for diagnosis of transplant
rejection. The practice of this invention can be useful to monitor
the acceptance of an organ transplant. All patients are given
immunosuppressants to prevent organ rejection and therefore it is
difficult to distinguish infection from rejection.
Moreover, this invention has utility for monitoring for metastatic
invasion. It has been found that tumor cells have different
patterns of enzymes from normal cells in the same tissue.
Identification of the types of enzymes is useful and important for
predicting metastatic potential and invasion. Tumor cells in
circulating blood can be useful to predict the progression of the
disease.
Preparation of Metabolically Active Whole Cells
The assay reagent is reacted with a metabolically active whole cell
analyte. The metabolically active whole cells are contained in
tissue, blood, cell cultures or other cells containing
constituents. Preferably, the metabolically active whole cells are
separated into cell types. The metabolically active cells to be
analyzed are isolated by known techniques such as differential
lysis, differential centrifugation, and affinity columns. However,
separation of the cells to be studied from other cells is not
always essential.
The cells are usually washed to remove any extracellular enzymes,
optionally with lysis or physical separation of unwanted cells.
Several preferred techniques for accomplishing this are summarized
in FIGS. 1A-1D.
The analysis of the segregated metabolically active cells provides
specificity for a particular enzyme analysis. For example, when the
metabolically active cell is a leukocyte blood cell, the method
comprises separating the leukocyte cell from the cell analyte,
washing the remaining leukocyte cell to remove any serum or plasma
enzymes, contacting an assay reagent compound with the leukocyte
cell, and determining fluorescence from the leukocyte cell (See
FIG. 1B). A modification of this method comprises washing the cell
analyte to remove any serum or plasma enzymes, contacting an assay
compound with the cell analyte, separating the leukocyte blood
cells from the cell analyte, and determining fluorescence from the
leukocyte cells (See FIG. 1A). In addition, another method that can
be used for cell analytes of leukocyte blood cells, nucleated
erythrocyte blood cells and platelets analytes comprises washing
the cell analyte to remove any serum or plasma enzymes, contacting
an assay compound with the analyte and determining fluorescence
from the analyte (See FIG. 1C).
To confirm that cells are metabolically active at the time of the
assay, it is desirable that the viability of the cells be checked
at the time of the assay. Several tests are useful to determine the
viability of cells. Trypan blue is a blue stain which diffuses into
the cell and is removed by cells if the cells are viable. Dead
cells will not remove the dye and will take on a blue color.
Propidium iodide is a DNA-RNA stain which, if the cell is dead and
membranes are damaged, will penetrate the cell and stain the
DNA-RNA. Fluorescein diacetate-propidium iodide will cause living
cells to take on a green color because the fluorescein diacetate
will be hydrolyzed, while dead cells become red from the propidium
iodide. Red blood cells do not undergo cell division, and therefore
a test for the presence of 2,3-diphosphoglucose dehydrogenase
(which is an indicator of cell division) is a useful test for
viability.
The assay of the present invention is particularly useful for
measuring intracellular concentrations of enzymes in mammalian
cells such as human cells. However, the assay should also be useful
in various or other types of cells which have metabolic
activity.
Assay Conditions
The concentration of the cells in the media should be high enough
to provide a reading of the desired number of cells within the
desired time period, taking into consideration the speed of the
instrument that is being used. For current flow cytometry
techniques, a concentration of about three million cells per
milliliter is appropriate to yield a measurement of about
10,000-15,000 cells in about 1-2 minutes.
The assay compound is generally employed in concentrations in
excess of the amount which can be completely hydrolyzed by the
quantity of enzyme within the time of the assay. An assay compound
concentration that is too high may have a negative affect on enzyme
activity, since the leaving group can be a negative feedback
inhibitor to enzyme activity.
The leaving group concentration in a cellular optimization is
determined using Km (a known rate constant) and VMA.sub.X (maximum
velocity) calculations. The leaving group is preferably present in
an amount from about 2 to about 100 x V.sub.MAX and most preferably
from about 2 to about 10 times the amount which can be completely
hydrolyzed by the enzyme within the duration of the assay period.
Preferred leaving group concentrations for particular enzymes are
included in Table 1.
The assay may be conducted either as a rate determination or as an
end point determination. Rate determinations are preferred, because
they are generally less affected by auto-fluorescence.
Consequently, a rate determination assay is more sensitive and
precise. In a rate determination, the fluorescence of the assay
compound-cell analyte mixture may be determined promptly after the
cell analyte is contacted with the assay compound. The ability to
see a signal and distinguish it from background noise determines
the initial starting point of data collection and the final data
point is preferably determined at the point where the slope of the
reaction rate changes, typically more than 2%.
Most cellular reactions do not strictly obey zero-order kinetics.
Most cellular enzymes show a delay between the time of exposure of
the cells to the assay compound, and the ability to detect a signal
that is greater than the background noise. Cellular enzymatic
reactions that do not obey zero order kinetics are still useful
measurements as first order, pseudo first order, or initial rate
measurements. Multiple enzymes in a reaction (mixed reactions) are
displayed by slope changes during the time course being
monitored.
In an endpoint determination, the enzyme hydrolysis reaction is
allowed to proceed for a predetermined length of time, usually at
V.sub.MAX. The reaction time can be calculated based on whether the
reaction is zero order or first order kinetics using
Michaelis--Menton methodology. Alternatively, the reaction time can
also be adjusted by a different elapsed time for pseudo-first order
reactions.
It has been determined that a number of factors will decrease the
reliability of the assay, and yield false positive, or erroneous
indications of enzymatic activity. These include (i) extended
reaction between the cell analyte and the assay compound; (ii)
another, non-targeted enzyme that is cleaving the leaving group;
(iii) auto-hydrolysis of the assay compound; (iv) inhibitors or
stimulators that are present and undetected; (v) cells that are no
longer metabolically active, or dead; (vi) mixed populations of
cells; (vii) a transfusion of the patient before sampling; (viii)
non-specific dye uptake by negative cells; and (ix) background
fluorescence. The creation of false negatives, or false indications
of a lack of enzymatic activity, can be caused by (i) insufficient
reaction between the cell analyte and the assay compound, (ii) a
hypoosmotic media leading to a decrease in cell activity; (iii) a
cell that is no longer metabolically active; (iv) burst cells; and
(v) the presence of inhibitors to the target enzyme.
It has been further determined that assays will be significantly
improved if reaction conditions are adjusted to maximize the
activity of the assayed enzyme relative to other non-assayed
enzymes which might otherwise compete for the leaving group. More
specifically, the targeted enzyme can be involved in a chain
cascade reaction of enzymes sequentially coupled to other enzymes,
as in a multi-enzyme reaction cascade.
The reaction conditions can be adjusted to maximize the efficiency
of the pathway, or to decrease the efficiency of competing
pathways. Such conditions preferably include at least one of pH,
choice of form of assay compound, temperature, osmotic pressure,
ionic strength, and reaction time.
The pH at which an enzyme is most efficient can be determined from
the literature, or determined empirically. As shown by FIG. 2, pH
maxima can have two peaks (optima). Therefore, the selection of the
appropriate pH should be made with care. In addition, care must be
exercised when using pH information from the literature, because
these values will usually be based on cytosol studies and not on
intact, metabolically active whole cells. Therefore, it is
preferable to use values from the literature only as a starting
point, and then to determine the appropriate pH from this reference
point. Generally, the pH will be between about 4.0 and 9.5. The pH
of the assay mixture is controlled by dissolving the cell analyte
and assay compound in an appropriate buffer. A list of preferred
pH's for particular enzymes is included in Table 1.
The form of assay compound can be important since some enzymes
require non-derivatized, natural structures for recognition of
binding and reaction, whereas other enzymes are less selective.
More specifically, derivatization and salt formation of the assay
compound are important properties for solubilization, enzyme
recognition and protection from auto-hydrolysis.
A reaction run using the same data collection window without the
enzyme source will determine auto-hydrolysis of the substrate and
therefore the potential for negative cells to absorb the dye
non-specifically resulting in false positive.
The time of the assay is typically less than 30 minutes, preferably
less than 20 minutes, usually between 5 seconds and 20 minutes, and
most preferably between about 10 seconds and about 5 minutes. Some
enzyme systems, such as esterase and phosphatase, can react with
the assay compound in shorter periods of time due to concentrations
of enzymes found in the cell. The reaction time should be limited
so that the effects of cellular expulsion of the indicator compound
will be avoided. Preferred time periods for assaying particular
enzymes are included in Table 1.
The temperature at which the assay is performed must be
physiologically acceptable to the cell. The temperature must be
high enough to retain viability and to ensure enzyme activity, but
not so high as to cause degradation or other deleterious reactions
involving the leaving group, the enzyme, or other components of the
mixture. Particular enzymes, or enzymes in particular pathways, are
more reactive at particular temperatures. The temperature is
preferably maintained between about 30.degree. C. to about
40.degree. C., more preferably between about 35.degree. C. and
about 38.degree. C., and most preferably between about 36.degree.
C. to about 38.degree. C. Preferred temperatures for a variety of
enzymes are shown in Table 1.
The osmotic pressure of the assay mixture is controlled to be
within physiological ranges from about 250 milliosmoles to 350
milliosmoles, preferably from about 275 milliosmoles to 320
milliosmoles. The osmotic pressure must be selected to maintain the
viability of the metabolically active whole cell. Variations in
osmotic pressures will result in lysis of the cell, severe
shrinking or shriveling (crenation) when too low, and swelling or
bursting (stomatolysing) of the cell when too high.
The ionic strength of the assay mixture should be selected so as to
avoid shriveling crenating or bursting (stromatolysing) of the
cells, and also to maximize the activity of the assayed enzyme
relative to other, non-assayed enzymes. An ionic strength that is
too low could deplete metals such as Ca.sup.+2, Mg.sup.+2, and
Zn.sup.+2, or cause insufficient amounts of anions such as
Cl.sup.-1, NO.sub.3.sup.-1, SO.sub.4.sup.-2 and PO.sub.4.sup.-3
which are the cofactors that can be used to improve enzymatic
activity. The ionic strength of the assay reagent is preferably
between about 0.1 to 0.3 .mu.. A list of preferred ionic strength
values for particular enzymes is included in Table 1.
The fluorescence reading is made after the reaction has occurred or
after a specific period of time. Typically, the reaction is stopped
by immersing the reaction container in ice and water which cools
the cells to about 0.degree. C. Sensing for one or more reaction
states by fluorescence determinations confirms cleavage of the
indicator group by the enzyme.
The fluorescence determinations are performed on a Image Analysis
System (IAS) or a Flow Cytometer (FC). The IAS is a microscope
based system that measures fluorescence known to those skilled in
the art. A representative example of an IAS is the Metamorph.TM. by
Universal Imaging Corporation, West Chester, Pa. The structure and
operation of flow cytometers is also well documented in the
literature. Alternatives to traditional FC include slit-scan FC and
stopped-flow FC. The type of instrument used to conduct the
experiments described in the examples was a flow cytometer (for
example, a Coulter Profile.RTM. flow cytometer manufactured by
Coulter Corporation of Miami, Fla). This flow cytometer measures
fluorescence across the entire cell. Flow cytometric methods which
measure fluorescence in only a part of the cell, such as slit scan
flow cytometry, have significant utility in the invention because
the background fluorescence is significantly reduced when
measurements are focused on the region of the cell where the enzyme
is located.
The fluorescence determinations can also be taken by a
spectrofluorometer which has the capability to measure the very low
fluorescence levels that are generated by the assay. The
spectrofluorometer is tuned to the excitation and emission
wavelengths of the particular indicator being used. Preferred
compounds such as rhodamine 110 and fluorescein have excitation and
emission wavelengths of about 495 to 498 nm (excitation) and 520 to
525 nm, respectively. The Model 8000C photon counting
spectrofluorometer manufactured by the SLM company, a subsidiary of
Milton Roy (Chicago, Ill.) was used.
The flow cytometer can perform additional measurements in addition
to a single wavelength fluorescence measurement. The flow
cytometers can be equipped to measure fluorescence at two or more
separate wavelengths. Such readings are useful to perform assays
according to the invention when using more than one assay compound,
or for using cell surface markers, such as monoclonal antibodies,
to determine cell morphology. Additional wavelengths are useful to
measure the activity of another enzyme, which can be a peptidase or
a different enzyme such as a phosphatase, saccharidase,
nucleotidase, esterase, or lipidase. Such additional tests are
useful for simultaneously characterizing disease states, and for
determining cell morphology and cell types.
Assay Protocols
Preferred sample preparations by which enzymes can be assayed using
the reagents prepared according to the method of the invention have
been developed. These sample preparations can be modified, and are
included herein to disclose those procedures that are currently
preferred.
The practice of the cell probe assay is divided into three parts:
1. Sample preparation, 2. Data Collection (i.e., Detection of
Fluorescence) and 3. Results (i.e., Data Analysis).
1. Sample Preparation:
Sample preparation can be divided into four different processes A,
B, C and D which are illustrated in FIGS. 1A, 1B, 1C and 1D,
respectively. The choice of sample preparation is dependent upon
the user and the analyte. The four processes are:
Process A: Examination of leukocytes or tissue cells with
erythrocyte contamination with post-lysing.
A sample, consisting of whole blood (in EDTA, Heparin or ACD) or
dissociated tissue or body fluids (synovial fluid) or cell culture
media is obtained and stored in a manner so as not to decrease
viability. The sample is washed sufficiently to remove plasma,
media, body fluid, debris and extra-cellular enzymes. The wash
media consists of a physiologically balanced buffered salt
solution. The washed cells are incubated at 37.degree. C. 50 .mu.L
of sample and 25 .mu.L of substrate media are mixed together and
allowed to incubate at 37.degree. C. for a predetermined amount of
time. At the end of the incubation period, unwanted cells are lysed
with a lytic reagent, i.e., erythrocytes are removed. Compatible
lytic systems are Q-Prep.TM., an acid lyse (formic acid/quench),
Erythrolyse.TM., (acid lyse/detergent/quench) or hypotonic ammonium
chloride. The sample is then measured for fluorescence. The
referenced lytic systems are commercially available from Coulter
Corporation, Miami, Fla.
Process B: Examination of leukocytes or tissue cells with
erythrocyte contamination with pre-lysing.
A sample, consisting of whole blood (in EDTA, Heparin or ACD) or
dissociated tissue or body fluids (synovial fluid) or cell culture
media is obtained and stored in a manner so as not to decrease
viability. Unwanted cells, i.e. erythrocytes, are lysed with a
lyric reagent. Compatible lyric systems are acid lysed (formic
acid/quench), IVCS lyse (quaternary ammonium salts)/quench or
hypotonic ammonium chloride. The sample is washed sufficiently to
remove plasma, media, body fluid, debris and extra-cellular
enzymes. The wash media consists of a physiologically balanced
buffered salt solution. The washed cells are incubated at
37.degree. C. 50 .mu.L of sample and 25 .mu.L of substrate media
are mixed together and allowed to incubate at 37.degree. C. for a
predetermined amount of time. At the end of the incubation period,
the sample is then measured for fluorescence.
Process C: Examination of platelets, erythrocytes, leukocytes,
dissociated tissue, body fluids and cell culture media.
A sample, consisting of whole blood (in EDTA, Heparin or ACD) or
dissociated tissue or body fluids (synovial fluid) or cell culture
media is obtained and stored in a manner so as not to decrease
viability. The sample is washed sufficiently to remove plasma,
media, body fluid, debris and extra-cellular enzymes. The wash
media consists of a physiologically balanced buffered salt
solution. The washed cells are incubated at 37.degree. C. 50 .mu.L
of sample and 25 .mu.L of substrate media are mixed together and
allowed to incubate at 37.degree. C. for a predetermined amount of
time. At the end of the incubation period, the sample is then
measured for fluorescence.
Process D: Examination of platelets, erythrocytes, leukocytes,
dissociated tissue, body fluids and cell culture media using a
mechanical separation to isolate a cell population.
A sample, consisting of whole blood (in EDTA, Heparin or ACD) or
dissociated tissue or body fluids (synovial fluid) or cell culture
media is obtained and stored in a manner so as not to decrease
viability. A mechanical separation to isolate a specific cell
population is performed, i.e., ficoll, differential centrifugation,
differential precipitation. The sample is washed sufficiently to
remove plasma, media, body fluid, debris and extra-cellular
enzymes. The wash media consists of a physiologically balanced
buffered salt solution. The washed cells are incubated at
37.degree. C. 50 .mu.L of sample and 25 .mu.L of substrate media
are mixed together and allowed to incubate at 37.degree. C. for a
predetermined amount of time. At the end of the incubation period,
the sample is then measured for fluorescence.
2. Detection of Fluorescence
The instruments used to detect fluorescence are the flow cytometer
or fluorescent microscope. There are four different instrument
configurations for the flow cytometer, A, B, C and D. Any of the
four configurations can be used with any one of the sample
preparations described above. The choice of which configuration is
selected is dependent upon the user and the information sought to
be obtained. The four configurations are:
Configuration A
Configuration A analyzes the cells by size, granularity and single
color. In the first configuration, the flow cytometer separates the
cells by size and granularity. The activity of an enzyme is then
assayed using the reagent compound. Two samples are allowed to
proceed at different times and the reaction is stopped. The
difference in fluorescence permits the calculation of a rate. Total
population counts preferred are 20,000 to 500,000 cells. Use of
light scatter or hematology parameters provide size and granularity
separation. Intensity bitmap of desired populations and
determination of fluorescent activity by single measurement point
or multi-point measurement can be employed. Determine count,
percentage and fluorescent intensity of a multi-modal population
representing enzymatic activity.
Configuration B
Configuration B analyzes the cells by size, granularity and two
colors. In the second configuration, the flow cytometer separates
the cells by size and granularity. Cell morphology is determined by
a fluorescence assay with a monoclonal antibody marker. The rate of
the hydrolysis of the assay compound is then determined. Total
population counts preferred are 20,000 to 500,000 cells. Use of
light scatter or hematology parameters provide size and granularity
separation. Intensity bitmap of desired populations and
determination of fluorescent activity by single measurement or
multi-point measurement can be employed. Determine count,
percentage and fluorescent intensity of a multi-modal population
representing enzymatic activity. The analysis is a 2-color analysis
measuring enzymatic activity in one color and surface-marker
antibody cell morphology in the other color.
Configuration C
Configuration C analyzes the cells by size, granularity, two colors
and backgate fluorescence. Configuration 3 is a modification of the
Duque method. Duque, R. E., "Flow Cytometric Analysis of Lymphomas
and Acute Leukemias", Annals of the New York Academy of Sciences,
Clinical Flow Cytometry, 677, pp. 309-325 (Mar. 20, 1993). The size
and granularity of the cell are separated by a flow cytometer using
light scatter and/or with surface markers, such as monoclonal
antibodies. A series of cell populations are determined, with
rearrangement of the histogram to identify the disease and normal
cells. The activity of the enzyme is then assayed. Total population
counts preferred are 20,000 to 500,000 cells. Use of light scatter
or hematology parameters provide size and granularity separation.
Intensity bitmap of desired populations and determination of
fluorescent activity by single measurement point or multi-point
measurement can be employed. Determine count, percentage and
fluorescent intensity of a multi-modal population representing
enzymatic activity. The analysis is a 2-color analysis measuring
enzymatic activity in one color and surface-marker antibody cell
morphology in the other color. Backgate fluorescence data on size
and granularity to determine count and percent of diseased
cells.
Configuration D
Configuration D analyzes activity of a population of cells over
time. Total population counts preferred are 20,000 to 500,000. Use
of light scatter or hematology parameters provide Size and
granularity separation. Intensity bitmap of desired populations and
determination of fluorescent activity by single measurement point
or multi-point measurement can be employed. Determine count,
percentage and fluorescent intensity of a multi-modal population
representing enzymatic activity. The analysis is a 2-color analysis
measuring enzymatic activity in one color and surface-marker
antibody cell morphology in the other color.
3. Data Analysis
The measured fluorescence intensity can be converted from mean
channel fluorescence (in peak or integrated mode) to MESF
(molecules of equivalent soluble fluorochrome, Flow Cytometry
Standards Corp., San Juan, Puerto Rico) or International Units of
hydrolysis per cell. A normal range of enzyme activity is
established by assaying males and females in sufficient quantity to
characterize the population levels statistically.
Various disease states are assayed for enzymatic activity and
compared to the normal range. Three conditions will exist from this
data:
1. Obvious increases or decreases in enzyme levels outside the
normal range
2. Patterns of enzyme activities representing morphology
3. Patterns of enzyme activities representing disease states
Artificial intelligence or Non-Negative Least Squares (NNLS)
programs and analysis of variance (ANOVA) programs are useful in
identifying patterns of enzyme activities representing morphology,
cell types and patterns of enzyme activities representing disease
states.
A first, and most obvious technique for disease diagnosis is
identifying the absence or presence of a single enzyme. One example
of such a single enzyme diagnosis is the diagnosis for Gaucher's
disease, which is diagnosed depending on the lack of a particular
enzyme, namely, glucocerebrosidase.
The remaining techniques treat the absence or presence of a
combination of enzymes as a complex interplay of metabolic systems,
wherein each cell contains a group of enzymes, and the
concentrations of these groups of enzymes are gaussian distributed,
having a normal range or values, wherein values in disease states
fall outside the normal range.
Analysis of Variance (ANOVA)
There are two steps in the mathematical analysis of the data. The
first step is to analyze the variance in the data. The purpose of
the analysis is to identify which combination of enzyme
concentrations for various cell types are diagnostic of particular
disease states or treatment modalities. (For simplicity, disease
states and treatment modalities will be referred to collectively as
disease states, henceforth).
To analyze the variance, each set of enzyme concentrations for each
enzyme measured for each cell type is considered as a component of
a composite measurement vector. A data matrix, such as the Full
Covariance Data Matrix in FIG. 14A, including columns of basis
measurement vectors known to characterize certain disease states is
generated. The rows of the matrix represent the measurement vector
components for each disease state under consideration. A variation
across a row indicates that the various disease states affect cell
metabolism such that the concentration of that enzyme in that cell
type is changed; obviously such a difference provides information
about the underlying disease state.
The data matrix needs to be developed for different disease states.
Patients for the disease state data matrix can be first identified
using conventional technologies. The disease state matrix can be
expanded to include differentiation of stages of the disease as
well as the influence of drug pharmokinetics on cellular function.
Separate studies of drug pharmokinetics on human tissue culture
cell lines can be performed to provide a reference.
The metabolism controlled by some enzymes in some cell types will
be insensitive to disease state. Others will react collectively in
a complex pattern with different enzymes in certain cells to
produce a pattern of enzyme concentrations that will definitively
characterize a particular disease state. The analysis of variance
is necessary to select out that combination of enzymes in
particular cells which are most useful in distinguishing among the
various disease states spanned by the basis measurement
vectors.
The selection of a relatively small number of components for the
measurement vector is necessary to simplify the later analysis,
reduce the number of physical measurements which must be made and
to reduce the effects of spurious noise generated from the
measurements and from individual variation among the same
population. An example of the selected number of components is
illustrated in FIG. 14A as the Reduced Covariance Data Matrix of
Strongly Contributing Factors.
Squared Deviations From the Mean
The simplest way to analyze the variance is to compute the variance
across the row for each row in the data matrix. Those rows with a
high variance correspond to enzymes whose concentration in a
particular cell type varies most strongly across the disease states
under consideration. This method of analysis neglects any
interaction of various enzymes with each other; however, it
provides a simple, gross indication of which enzymes in which cell
types are most affected by the disease state. This technique was
used in the "Variance" column of Table 5.
Eigenvector or Principal Components Analysis of Variance
A more complete way to analyze variance is to compute the
eigenvalues and eigenvectors of the data matrix, as described for
example in J. D. Jobson, Applied Multivariate Data Analysis,
Springer Verlag N.Y. (1992). Such an analysis is conventionally
termed an eigenvector analysis or a principal components analysis
(PCA). In practice, rather than on the data matrix, D, itself, the
eigenvector analysis is performed on the covariance matrix of the
data (D-m).sup.t (D-m), where the superscript t refers to the
transpose of the matrix and m is the vector whose components are
the mean of each row of D. Each resulting eigenvector includes a
particular weighted combination of the measurement vector
components which act in concert with each other. Each eigenvector
has a corresponding eigenvalue which is proportional to the total
variance in the measurement which is accounted for by that
eigenvector. Each eigenvector is also orthogonal or independent of
every other eigenvector. This technique was used in the
"Eigenvector 1" and "Eigenvector 2" columns of Table 5.
For the data matrices here, if the eigenvectors are arranged in
order of decreasing eigenvalue, the vast majority of the variance
is accounted for by the first several eigenvectors. Thus, the
principal factors distinguishing the various disease states under
consideration can be captured with a small number of combinations
of the enzyme concentrations in the various cell types. FIG. 14A
illustrates the process of developing the reduced covariance data
matrix by eigenvector analysis.
An examination of the combination of measurement vector components
contributing most to distinguishing different disease states, in
light of the metabolic pathways linking the various enzymes, can be
used to understand the underlying metabolic changes occurring with
the various disease states. The foregoing eigenvector analysis is
used to select from the entire series of measured enzyme
concentrations for different cell types, the combination of enzymes
which is most useful in characterizing the disease state.
Diagnosing Disease State, Based on Measurement NNLS
Once the measurement vector and data matrix are reformed with the
selected (reduced number of) enzyme concentrations for particular
cell types, measurements of patients with unknown diseases
(presumed to be within the basis disease states of the data matrix)
can be used to diagnose their disease state. Two methods are
described here to accomplish the second step of the mathematical
analysis of the data, the inference of disease state from the
measurement vector: a Non-Negative Least Squares based algorithm
(NNLS) and a neural net.
NNLS is a non-negatively constrained least squares solution to the
problem of determining the disease state from the reduced
measurement vector. The algorithm, which is disclosed in C. L.
Lawson and R. J. Hanson, Solving Least Squares Problems,
Prentice-Hall N.J. (1974) finds that linear combination of the
basis measurement vectors which most closely fits, in a least
squares sense, the measurement vector of the patient whose state is
being diagnosed. The resulting solution is a vector the magnitude
of whose components reflect the probability that the unknown
disease state is each one of the basis disease states. The
algorithm constrains the components of the solution vector to be
non-negative; this constraint can be applied because the components
of the vector represent probabilities, which by definition must be
non-negative. The non-negativity constraint is extremely important
in stabilizing the solution to this often mathematically
ill-conditioned inversion problem.
Ideally, the solution vector has only a single non-zero component,
in this case, the disease state corresponding to the chosen basis
measurement vector is the diagnosis of the patient. Because of
noise in the measurements and individual biological variation among
individuals or bemuse a patient is afflicted with more than one
disease at a time, the solution vector provides a range of
possibilities for the diagnosis; the component with the highest
magnitude representing the most probable disease state, etc. FIG.
14B illustrates the process of extracting predicted disease state
probabilities from the reduced covariance data matrix.
As illustrated in FIG. 14A, a database is obtained for both normal
states and various disease states. An example of an array of values
obtained for 5 cell types (lymphocytes, monocytes, platelets,
granulocytes and erythrocytes) using 50 different enzyme assays is
shown in Table 3A-3C. This Table further gives a mean value, two
standard deviation low value and two standard deviation high value
for each cell for each enzyme.
TABLE 3A
__________________________________________________________________________
NORMAL LEUKOCYTE ENZYME ACTIVITY LYMPHS MONOS GRANS Mean Lo Hi Mean
Lo Hi Mean Lo Hi
__________________________________________________________________________
Aminopep LEU 34.94 14.73 55.14 120.66 35.52 205.80 80.19 28.42
131.96 Aminopep M ALA 75.85 47.46 104.25 172.33 107.51 237.15
149.02 85.05 212.98 Pro Aminopep PRO 1.70 0.53 2.88 6.83 2.62 11.04
9.19 3.96 14.41 Aminopep M LYS 1.16 0.33 1.99 7.56 3.83 11.29 4.27
0.00 8.88 Aminopep M, N GLY 18.18 7.21 29.14 81.47 30.13 92.82
29.48 9.82 49.14 Aminopep N SER 1.35 0.28 2.41 6.49 2.71 10.26 3.40
0.75 6.06 Endopeptidase I Z-ARG 2.02 0.67 3.38 16.80 7.83 25.78
7.22 0.37 14.06 Endopeptidase I ARG 1.47 0.50 2.45 10.34 5.13 15.54
5.16 0.23 10.10 Aminopep A ASP 0.26 0.04 0.49 0.66 0.06 1.27 0.45
0.01 0.90 Cathepsin B VS 3.81 0.57 7.06 19.61 2.86 36.36 8.27 2.68
13.87 Cathepsin B VS-M 3.78 0.86 6.70 31.14 5.17 57.11 7.07 1.75
12.40 Cathepsin B VK 1.87 0.05 3.69 20.38 0.00 54.92 5.10 0.65 9.54
Cathepsin B VK-M 2.05 0.00 4.40 43.37 6.71 80.04 4.31 0.00 8.76
Cathepsin B QS 1.70 1.06 2.34 10.66 4.02 17.30 4.67 2.08 7.25
Cathepsin B QS-M 1.14 0.67 1.61 8.69 2.81 14.57 2.71 1.43 4.00
Cathepsin B LG 13.83 6.56 21.09 44.56 22.50 66.62 29.99 11.28 48.69
Cathepsin B LG-M 4.71 1.33 8.08 23.92 0.00 48.54 10.54 3.83 17.26
Dipep Peptidase II KA 11.47 3.00 19.93 39.35 16.82 61.88 29.15 9.25
49.05 Dipep Peptidase II KA-M 0.55 0.30 0.80 9.74 1.47 16.01 1.74
0.82 2.65 Dipep Peptidase IV Z-AA 151.20 113.68 188.73 328.25
243.23 413.27 363.20 273.63 452.87 Dipep Peptidase IV Z-AA-M 94.47
47.80 141.14 210.71 112.01 309.42 252.24 138.76 365.72 Dipep
Peptidase IV Z-GP 107.56 72.84 142.28 225.10 155.83 294.37 240.99
148.98 332.99 Dipep Peptidase IV Z-GP-M 53.74 29.05 78.44 119.81
70.14 169.48 130.62 63.36 197.87 Cathepsin D GL 9.42 3.84 15.00
41.78 20.02 63.54 29.16 11.99 46.33 Cathepsin D GL-M 2.56 0.00 5.34
89.11 0.00 195.37
12.01 0.00 27.36 Cathepsin C Z-AG 15.17 7.48 22.86 34.51 18.95
50.08 33.19 17.20 49.18 Cathepsin C Z-AG-M 14.24 2.97 25.52 37.75
12.88 62.61 34.70 9.31 60.09 Dipep Peptidase IV AA-TFA 56.95 3.01
110.89 158.31 31.98 284.63 154.81 25.75 283.87 Dipep Peptidase IV
AA-M 9.88 0.00 24.56 58.29 1.79 114.79 29.91 9.29 50.53 Cathepsin D
Z-TP 6.5 21.97 8.98 34.96 49.69 22.25 77.13 58.05 24.15 91.96
Cthepsin D Z-TP 6.5-M 20.62 9.03 32.22 45.70 21.61 69.90 55.07
23.22 86.91 Cathepsin B LLR 4.09 0.00 9.90 54.32 28.31 80.32 9.46
0.00 23.73 Cathepsin B LLR-M 3.38 0.50 6.26 62.85 14.37 111.33 7.58
0.00 24.34 Cathepsin B LGLG 1.42 0.76 2.08 11.36 0.00 23.64 4.08
1.93 6.23 Cathepsin B LGLG-M 1.34 0.38 2.29 40.74 0.00 98.16 3.24
1.12 5.37 Estertase FDA 13.38 0.00 27.21 66.25 17.06 115.43 68.37
12.90 123.84 Monocytic Esterase FDA-NAF 17.98 0.43 35.54 93.36 0.00
210.77 81.99 17.39 146.60 Peroxidase DCFH-DA MES 4.11 0.68 7.53
12.28 2.23 22.34 7.49 0.95 14.04 Activated Peroxidase DCFH MES PMA
4.69 0.98 8.41 22.18 0.00 45.35 11.02 0.00 28.46 Collagenase GPLGP
8.97 3.64 14.30 23.38 10.98 35.79 16.88 6.80 26.96 Collagenase
GPLGP-M 7.89 2.40 13.37 23.32 10.36 36.28 13.82 4.39 23.24
Collagenase GPGA 0.36 0.22 0.60 1.01 0.01 2.01 1.00 0.36 1.63
Elastase RGES 1.43 0.75 2.12 8.37 4.28 12.46 3.96 2.09 5.83
Glucosidase DIGLUC 0.94 0.42 1.46 6.58 0.25 10.91 2.35 0.10 4.60
Acid Phosphatase PO4 5.0 54.29 17.08 91.49 148.32 61.42 235.23
85.42 13.42 157.42 Galactidase DIGALAC 0.38 0.00 1.05 5.94 0.00
17.90 1.58 0.00 4.32 Cathepsin C Z-TP 8.7-M 30.99 16.24 45.75 71.45
38.94 103.97 80.16 45.97 114.35 Cathepsin C Z-TP 8.7 35.01 24.75
45.26 76.00 53.89 98.11 82.35 59.36 105.33 Glucosidase DIGLUC (FL)
0.23 0.15 0.32 1.89 0.00 4.55 0.45 0.07 0.83 Neutral Butyrate DIBUT
7.5 Acidic Butyrate DIBUT 6.5 4.08 0.00 12.16
10.37 0.00 30.20 6.43 0.00 18.24 Esterase CHLOAC 2.49 0.85 4.13
7.33 1.76 12.89 6.48 1.40 11.57 Esterase DIACET 6.5 109.89 39.37
180.41 534.00 322.46 745.54 408.88 251.29 566.46 Esterase DIACET
7.5 89.30 0.00 219.98 432.78 0.00 986.33 348.01 0.00 781.34 Acidic
Prop Esterase DIPRO 6.5 86.11 0.00 216.26 441.65 0.00 1040.17
349.18 0.00 838.78 Neutral Prop Esterase DIPRO 7.6 95.34 0.00
219.08 489.27 0.00 1106.82 429.17 0.00 977.31 Acidic Valerate
Esterase DIVAL 6.5 33.13 20.42 45.84 184.51 119.00 250.02 92.81
47.67 137.95 Acidic Hex Esterase DIHEX 6.5 15.10 4.22 25.97 72.87
24.73 121.00 33.84 11.39 56.29 Neutral Hex Esterase DIHEX 7.5 7.54
0.00 15.15 37.22 10.06 64.37 17.57 2.89 32.24 Acidic Hep Esterase
DIHEP 6.5 9.17 0.00 19.35 32.25 3.51 60.99 18.57 3.86 33.28 Neutral
Hep Esterase DIHEP 7.5 9.73 0.00 19.52 36.15 7.40 64.89 20.41 0.92
39.89 Acidic Pal Esterase DIPAL 6.5 0.15 0.00 0.31 0.30 0.16 0.45
0.14 0.09 0.20 Neutral Pal Esterase DIPAL 7.5 0.14 0.00 0.29 0.30
0.13 0.47 0.14 0.10 0.19
__________________________________________________________________________
TABLE 3B ______________________________________ NORMAL ERYTHROCYTE
ENZYME ACTIVITY SUBSTRATE ENZYME MEAN 2SD LO 2SD HI
______________________________________ LEU Aminopeptidase 0.107
0.105 0.108 ALA Aminopeptidase M 0.237 0.000 0.602 PRO Pro
Aminopeptidase 0.121 0.087 0.155 LYS Aminopeptidase M 0.109 0.099
0.118 GLY Aminopeptidase M, N 0.163 0.025 0.300 SER Aminopeptidase
N 0.107 0.107 0.107 Z-ARG Enodpeptidase I 0.106 0.106 0.106 ARG
Endopeptidase I 0.106 0.104 0.107 ASP Aminopeptidase A 0.106 0.106
0.106 VS Cathepsin B 0.138 0.073 0.203 VS-M Cathepsin B 0.176 0.027
0.324 VK Cathepsin B 0.170 0.097 0.242 VK-M Cathepsin B 0.129 0.093
0.164 QS Cathepsin B 0.121 0.108 0.133 QS-M Cathepsin B 0.116 0.113
0.119 LG Cathepsin B 0.138 0.080 0.195 LG-M Cathepsin B 0.131 0.129
0.132 KA Dipeptidylpeptidase II 0.118 0.096 0.139 KA-M
Dipeptidylpeptidase II 0.110 0.104 0.116 Z-AA Dipeptidylpeptidase
IV 0.484 0.000 1.454 Z-AA-M Dipeptidylpeptidase IV 0.411 0.000
1.227 Z-GP Dipeptidylpeptidase IV 0.438 0.000 0.976 Z-GP-M
Dipeptidylpeptidase IV 0.334 0.000 0.818 GL Cathepsin D 0.190 0.111
0.269 GL-M Cathepsin D 0.286 0.000 0.734 Z-AG Cathepsin C 0.225
0.000 0.482 Z-AG-M Cathepsin C 0.171 0.115 0.226 AA-TFA
Dipeptidylpeptidase IV 0.144 0.070 0.218 AA-M Dipeptidylpeptidase
IV 0.131 0.097 0.165 Z-TP 6.5 Cathepsin C 0.132 0.098 0.166 Z-TP
6.5M Cathepsin C 0.168 0.070 0.265 LLR Cathepsin B 0.225 0.000
0.529 LLR-M Cathepsin B 0.476 0.000 1.492 LGLG Cathepsin B 0.122
0.091 0.153 LGLG-M Cathepsin B 0.150 0.059 0.241 FDA Esterase 0.126
0.089 0.163 FDA-NAF Monocytic Esterase 0.136 0.088 0.184 DCFH-DAMES
Peroxidase 0.286 0.000 0.764 DCHFMESPMA Activated peroxidase 0.268
0.000 0.693 GPLGP Collagenase 0.164 0.136 0.192 GPLGP-M Collagenase
0.119 0.103 0.134 GFGA Collagenase 0.131 0.073 0.188 RGES Elastase
0.118 0.098 0.138 DGLUC 49 Glucosidase 0.114 0.109 0.118 DPO4 46
Acid Phosphatase 0.151 0.090 0.211 GALAC 50 Galactidase 0.119 0.111
0.127 TP 8.7M 48 Cathepsin C 0.212 0.000 0.467 TP 8.7 47 Cathepsin
C 0.161 0.054 0.267 FL GLUC Glucosidase 0.110 0.110 0.110
______________________________________
TABLE 3C ______________________________________ NORMAL PLATELET
ENZYME ACTIVITY SUBSTRATE ENZYME MEAN 2SD LO 2SD HI
______________________________________ LEU Aminopeptidase 1.006
0.640 2.156 ALA Aminopeptidase M 1.726 0.846 3.422 PRO Pro
Aminopeptidase 0.295 0.111 0.854 LYS Aminopeptidase M 0.232 0.110
0.440 GLY Aminopeptidase M, N 3.622 0.897 8.030 SER Aminopeptidase
N 0.309 0.135 0.579 Z-ARG Enodpeptidase I 0.262 0.133 0.448 ARG
Endopeptidase I 0.268 0.128 0.499 ASP Aminopeptidase A 0.403 0.170
1.274 VS Cathepsin B 0.576 0.175 2.007 VS-M Cathepsin B 0.592 0.180
1.622 VK Cathepsin B 0.839 0.144 3.080 VK-M Cathepsin B 0.486 0.153
1.343 QS Cathepsin B 0.481 0.186 1.176 QS-M Cathepsin B 0.499 0.280
1.194 LG Cathepsin B 0.470 0.182 0.922 LG-M Cathepsin B 0.438 0.143
1.032 KA Dipeptidylpeptidase II 0.447 0.161 1.034 KA-M
Dipeptidylpeptidase II 0.277 0.126 0.562 Z-AA Dipeptidylpeptidase
IV 1.372 0.480 3.438 Z-AA-M Dipeptidylpeptidase IV 1.199 0.545
3.253 Z-GP Dipeptidylpeptidase IV 2.668 0.864 5.867 Z-GP-M
Dipeptidylpeptidase IV 1.415 0.481 3.883 GL Cathepsin D 0.702 2.010
1.930 GL-M Cathepsin D 0.511 0.254 1.235 Z-AG Cathepsin C 7.900
0.411 21.322 Z-AG-M Cathepsin C 5.113 0.280 13.295 AA-TFA
Dipeptidylpeptidase IV 0.622 0.218 1.274 AA-M Dipeptidylpeptidase
IV 0.350 0.145 0.684 Z-TP 6.5 Cathepsin C 2.105 0.251 4.841 Z-TP
6.5M Cathepsin C 2.348 0.347 5.889 LLR Cathepsin B 2.150 0.484
5.562 LLR-M Cathepsin B 0.434 0.239 0.886 LGLG Cathepsin B 2.275
0.230 10.086 LGLG-M Cathepsin B 4.013 0.211 9.648 FDA Esterase
15.353 2.960 29.955 FDA-NAF Monocytic Esterase 7.201 2.310 14.369
DCFH-DAMES Peroxidase 0.764 0.317 1.840 DCFH-DAATRIS Super oxide
dismutase 3.556 1.640 7.982 DCHFMESPMA Activated peroxidase 1.979
0.576 7.045 DCFHTRISPMA Activated super oxide 5.111 1.870 12.045
dismutase ______________________________________
Artificial Intelligence By Back Propagation (Neural Net)
An alternate method of analyzing the data is via neural net.
To determine the interrelationship of enzyme function in both the
cell and cell type, the ratios of enzyme activities needs to be
analyzed. To fully analyze all possible combinations of a data set,
an artificial intelligence system such as "Neuroshell.TM." (Ward
Systems Group Inc., Frederick, Md.) may be used.
The basic building block of artificial intelligence neural network
technology is the simulated neuron, which processes a number of
inputs to produce an output. Inputs and outputs are numeric values
between 0 and 1 which represents positive stimulation close to 1
and negative stimulation close to 0. Inputs are data entered and
outputs either come from other neurons or are displayed as results.
The process by which the neuron processes its inputs to arrive at
an output is usually a summation of inputs followed by a linear
function applied to the sum. Independent neurons are of little use
unless connected to a network of neurons called nodes. Nodes are
layered and interconnected to receive information from each other.
As each input node passes information to each other and the next
layer, the values are weighed to represent the connection strength.
To positively reinforce a connection the weight is raised and
likewise to negatively reinforce or inhibit a connection the weight
is lowered. The network processes data by accepting input patterns
into input nodes or Defining Characteristics. The network produces
output patterns which are called Classifying Characteristics. The
user of the algorithm can adjust the output pattern by adjusting
output thresholds. Feedback from the user determines whether the
reinforcement is positive or negative. Learning in a neural network
occurs when a set of input patterns (cell type and enzyme
concentration) is given with a known output pattern (Disease state
or Normal). This is called a sample case. The error between the
predicted and actual outputs for a given output node is measured
and the total error is one-half of the sum of the squares of the
difference. The weights leading to this output node are modified
slightly (specified by the user as learning rate, a percentage of
the error to be used in the next iteration) during each iteration
of a learning session in the direction required to produce a
smaller error the next time the same pattern is presented. This is
how the neural network "learns". Learning is continued until the
consummate error of all output nodes falls below a learning
threshold controlled by the user. Upon completion of learning, the
network should be capable of reproducing the correct output pattern
(disease or normal) when presented with one of the input patterns
it has learned. Moreover, the network is capable of generalizing by
recognizing an input pattern close to a pattern it has learned and
produces an output close to a pattern it was trained to produce. A
simple two layered network is incapable of learning complex
patterns. Back propagation uses one or more layers of hidden nodes
and a nonlinear function algorithm The weight applied to the nodes
must then be back propagated through all layers of the nodes. The
number of hidden nodes is determined by the user for a specific
problem. If too few hidden nodes are used, then all the unique
situations found in the sample case will not be explored and if too
many are used, learning will never complete. The neural network
approach provides the opportunity to look at ratios of cell types
and enzyme levels in all possible combinations for both disease and
health using a simple format.
Progression of a disease during treatment to monitor "return to
normalcy" or further increase in stage or complication with
additional disease states can be done by monitoring the NNLS
predictive disease probabilities over time or the value of the
Neural Network score as it approaches normalcy or the
three-dimensional plotting of cell-type enzyme activity patterns
comparison to normal. Recurrent Neural Networks may also be used
for time series data. Examples of these types are the Probabilistic
Neural Network (PNN), General Regression Neural Network (GRNN) and
the Kohonen-Realty Neural Network.
One embodiment of an analysis system using neural networks is
illustrated in FIG. 15. The same database illustrated in FIG. 14A
using the ANOVA technique is also used in the neural network
implementation. The database is input into this neural network has
a training set and the user sets a threshold, learning rate, and
learning momentum. Once the neural network has "learned" on the
plurality of sample cases, a test case is input to the neural
network including an unknown to be classified as a disease state or
normal state. Based on what the neural network as learned with a
plurality of sample cases, the neural network outputs the best
predictor of the test case. The neural network may also be utilized
to perform time course monitoring of an individual patient for
return to normalcy, using an advanced probabilistic neural network
(TNN) program.
Look-Up Tables
An alternative to the NNLS and Neural Net analyses described above
uses Look-Up tables and is similar to the Expert System described
in "White Cell and Thrombocyte Disorders--Standardized
Self-learning Flow Cytometric List Mode Data Classification with
the CLASSIF1 Program System", Valet et al, Ann. N.Y. Acad. Sci.,
677: 233-251 (1993).
Definitions
As used herein, either individually or as part of a larger group,
"alkyl" means a linear, cyclic, or branched-chain aliphatic moiety
of one to 10 carbon atoms; "substituted alkyl" means an alkyl group
having a substituent containing a heteroatom or heteroatoms such as
N, O, or S; "aryl" means an aromatic moiety, e.g., phenyl, of 6 to
18 carbon atoms, unsubstituted or substituted with one or more
alkyl, substituted alkyl, nitro, alkoxy, or halo groups; and
"alkaryl" means an aryl moiety of 7 to 19 carbons having an
aliphatic substituent, and optionally, other substituents such as
one or more alkyl, substituted alkyl, alkoxy or amino groups.
"Aralkyl" means a linear or branched-chain aliphatic moiety of six
to 18 carbon atoms comprising an aryl group or groups.
The following common chemical abbreviations are used in the
examples:
t-BOC=tertiarybutyloxycarbonyl
EDAC=1-ethyl-3-(3'-dimethylaminopropylcarbodiimide)-hydrochloride
FMOC=9-fluorenylmethyloxycarbonyl
BOP=benzotriazoly-N-oxy-tris(dimethylamino)-phosphonium-hexafluorophosphate
HBOT=1-hydroxybenzotriazole
HPLC=High pressure liquid chromatography
TLC=Thin layer chromatography
V:V=Volume to volume
The amino acids are abbreviated as follows:
______________________________________ Amino Acid Abbreviation
______________________________________ L-alanine Ala or A
L-arginine Arg or R L-asparagine Asn or N L-aspartic acid Asp or D
L-cysteine Cys or C L-glutamic acid Glu or E L-glutamine Gln or Q
glycine Gly or G L-histidine His or H L-isoleucine Ile or I
L-leucine Leu or L L-lysine Lys or K L-methionine Met or M
L-phenylalanine Phe or F L-proline Pro or P L-serine Ser or S
L-threonine Thr or T L-tryptophan Trp or W L-tyrosine Tyr or X
L-valine Val or V ______________________________________
The synthesis of the assay compounds can be further understood by
reference to the Examples. It will be appreciated, however, that
the invention is not limited to the described examples, and that
other methods of preparation could be suitable to prepare reagents
according to the invention.
EXAMPLE 1
Preparation of Monopeptide Derivative of Rhodamine 110 Employing
the EDAC Procedure
A 10-fold excess of a FMOC amino acid is placed into a round bottom
flask containing a 50:50 pyridine-dimethylformamide solution (V:V)
and stirred until complete solution occurs. To this stirred
solution is added a 12-fold excess of EDAC and the admixture is
stirred for 30 minutes. A solution of rhodamine 110 dissolved in a
minimum of a 50:50 pyridine-dimethylformamide (V:V) is added
dropwise to the reaction solution. This addition requires 15-20
minutes and the reaction solution is allowed to stir at room
temperature overnight. The solution is concentrated under reduced
pressure to an oil. This oil is dissolved into an appropriated
organic solvent and the product is purified by normal phase HPLC,
using solvents of increasing polarity (methylene chloride, 1%
methanol-chloroform, 2% methanol-chloroform, etc.). The eluate
containing the product is concentrated under reduced pressure
affording a crystalline material and the purity and identity are
checked by analytical reverse phase high pressure liquid
chromatography and thin layer chromatography.
The crystalline material is treated with a 5% solution of
piperidine, dissolved in dimethylformamide. The reaction is stirred
for 45 minutes and concentrated under reduced pressure. The
resulting solid is triturated several times with pentane and then
dissolved in a minimum of methanol and a 5-fold excess of
trifluoroacetic acid is added. The solution is concentrated under
reduced pressure to dryness and the resulting solid is centrifuged
with cold diethyl ether until the ether triturate has a pH=7. If
the monopeptide is polar, then the remaining protective group is
removed by treating with a 30 to 50% trifluoroacetic acid solution
in methylene chloride for four hours at room temperature. The
solution is concentrated under reduced pressure to dryness and the
resulting solid is centrifuged with cold diethyl ether until the
ether triturate has a pH=7. A final purification of this
trifluoroacetic acid substrate is effected with reverse phase EPLC,
using solvents of decreasing polarity (water, acetonitrile,
trifluoroacetic acid). The eluate containing the product is
concentrated under reduced pressure and the aqueous solution is
lyophilized. The product's purity and identity are checked by
analytical reverse phase high pressure liquid chromatography, thin
layer chromatography, and photon counting spectrofluorometry. The
purity and stability of the product are also measured by monitoring
the background fluorescence, autohydrolysis and enzymatic activity
using the product as a substrate after storage of the product at
4.degree. C. FIGS. 10A and 10B illustrate the stability and purity
of a monopeptide-TFA salt derivative Proline-rhodamine 110 which
was prepared by the procedure described in this Example. Stability
(background fluorescence) is shown in FIG. 10A. Autohydrolysis
(diamonds) and enzyme rate (squares) are shown in FIG. 10B.
EXAMPLE 2
Preparation of Dipeptide Derivative of Rhodamine 110 Employing the
EDAC Procedure
A 6-fold excess of the FMOC amino acid is placed into a round
bottom flask containing a 50:50 pyridine-dimethylformamide solution
(V:V) and stirred several minutes. To this well-stirred solution is
added a 12-fold excess of EDAC and the admixture is stirred an
additional 30 minutes. A solution of the monopeptide of rhodamine
110 dissolved in a minimum of 50:50 pyridine-dimethylformamide
solution (V:V) is added dropwise over a period of 15 to 20 minutes.
The reaction is stirred at room temperature for 16 hours and then
concentrated to an oil under reduced pressure. This oil is
dissolved in a minimum of an organic solvent and the crude product
is purified by normal phase HPLC. The eluate containing the desired
product is collected and concentrated under reduced pressure
affording a crystalline material and the purity and identity are
checked by analytical reverse phase high pressure liquid
chromatography and thin layer chromatography. The FMOC blocking
group is removed by dissolving the solid in a 5%
piperidine-dimethylformamide solution and stirred at room
temperature for one hour. The solution is concentrated to dryness
under reduced pressure. The resulting solid is triturated several
times with pentane to remove the FMOC polymer. The solid is
dissolved in a minimum of methanol and a 5-fold excess of
trifluoroacetic acid is added. The solution is concentrated to
dryness and the resulting solid is centrifuged with cold diethyl
ether until the ether triturate has a pH=7. If the dipeptide is
polar, then the remaining protective group(s) is removed by
treating with a 30 to 50% trifluoroacetic acid solution in
methylene chloride for four hours at room temperature. The solution
is concentrated under reduced pressure to dryness and the resulting
solid is centrifuged with cold diethyl ether until the ether
triturate has a pH=7. A final purification of this trifluoroacetic
acid substrate is effected with reverse phase HPLC. The eluate
containing the product is concentrated under reduced pressure and
the aqueous solution is lyophilized. The product's purity and
identity are checked by analytical reverse phase high pressure
liquid chromatography, thin layer chromatography and photon
counting spectrofluorometry. The purity and stability of the
product are also measured by monitoring the background
fluorescence, autohydrolysis and enzymatic activity using the
product as a substrate after storage of the product at FIGS.
11A-11F illustrate the stability and purity of the TFA salts of
several dipeptide derivatives of rhodamine 110 prepared by the
procedure described in this Example. The Figures describe the
following:
FIG. 11A (background fluorescence, Val-Lys.cndot.TFA);
FIG. 11B (autohydrolysis and enzyme rate, Val-Lys.cndot.TFA);
FIG. 11C (background fluorescence, Val-Ser.cndot.TFA);
FIG. 11D (autohydrolysis and enzyme rate, Val-Ser.cndot.TFA);
FIG. 11E (background fluorescence, Leu-Gly.cndot.TFA); and
FIG. 11F (autohydrolysis and enzyme rate, Leu-Gly.cndot.TFA).
EXAMPLE 3
Preparation of Polypeptide Derivative of Rhodamine 110 Employing
the EDAC Procedure
A 6-fold excess of the FMOC polyamino acid is placed into a round
bottom flask containing a 50:50 pyridine-dimethylformamide solution
(V:V) and stirred until solution occurs. To this well stirred
solution is added a 12-fold excess of EDAC and the admixture is
stirred an additional 30 minutes. A solution of the monopeptide of
rhodamine 110 dissolved in a minimum of 50:50
pyridine-dimethylformamide solution (V:V) is added dropwise over a
period of 15 to 20 minutes. The reaction is stirred at room
temperature for 16 hours and then concentrated to an oil under
reduced pressure. The oil is dissolved in a minimum of an organic
solvent and the crude product is purified by normal phase HPLC. The
eluate containing the desired product is collected and concentrated
under reduced pressure affording a crystalline material and the
purity and identity are checked by analytical reverse phase high
pressure liquid chromatography and thin layer chromatography. The
FMOC blocking group is removed by dissolving the solid in a 5%
piperidine dimethylformamide solution and stirred at room
temperature for one hour. The solution is concentrated under
reduced pressure to dryness under reduced pressure. The resulting
solid is triturated several times with pentane to remove the FMOC
polymer. The solid is dissolved in a minimum of methanol and a
5-fold excess of trifluoroacetic acid is added. The solution is
concentrated under reduced pressure to dryness and the resulting
solid is centrifuged with cold diethyl ether until the ether
triturate has a pH=7. If the polypeptide is polar then the
remaining group(s) is removed by treating with a 30 to 50%
trifluoroacetic acid solution for four hours at room temperature.
The solution is concentrated under reduced pressure to dryness, and
the resulting solid is centrifuged with cold diethyl ether until
the ether triturate has a pH=7. A final purification of this
trifluoroacetic acid substrate is effected with reverse phase HPLC.
The eluate containing the product is concentrated under reduced
pressure and the aqueous solution is lyophilized. The product's
purity and identity are checked by analytical reverse phase high
pressure liquid chromatography, thin layer chromatography and
photon counting spectrofluorometry.
EXAMPLE 4
Preparation of a Dipeptide Derivative of Rhodemine 110 Employing
the HOBT-BOP Procedure
A 4-fold excess of the FMOC amino acid, and a 4-fold excess of EOBT
and BOP are placed into a round bottom flask containing a 0.6
millimolar solution of N-methylmorpholine in dimethylformamide and
stirred for 10-15 minutes. To this solution is added dropwise a
solution of the monopeptide of rhodamine 110 dissolved in a minimum
amount of a 0.6 millimolar solution of N-methylmorpholine in
dimethylformamide. This addition requires 5-10 minutes, and the
reaction is stirred at room temperature for four hours. The
reaction solution is concentrated under reduced pressure to an oil.
This oil is dissolved in methylene chloride and the crude product
is purified by normal phase HPLC. The eluate containing the desired
product is collected and concentrated under reduced pressure
affording a crystalline material. The purity and identify of this
material are checked by analytical reverse phase HPLC and thin
layer chromatography. The FMOC blocking is removed by dissolving
the solid in a 5% piperidine-dimethylformamide solution and stirred
at room temperature for one hour. The solution is concentrated
under reduced pressure, and the resulting solid is triturated
several times with pentane to remove the FMOC polymer. The
remaining solid is dissolved in a minimum of methanol and a 5-fold
excess of trifluoroacetic acid is added. The solution is
concentrated under reduced pressure and the resulting solid is
centrifuged with cold diethyl ether until the ether triturate has a
pH=7. If the dipeptide is polar then the remaining protective
group(s) is removed by treating with a 30 to 50% trifluoroacetic
acid solution in methylene chloride for four hours at room
temperature. The solution is concentrated under reduced pressure to
dryness, and the resulting solid is centrifuged with cold diethyl
ether until the ether triturate has a pH=7. A final purification of
this trifluoroacetic acid substrate is effected with reverse phase
HPLC. The eluate containing the product is concentrated under
reduced pressure and the aqueous solution is lyophilized. The
product's purity and identity are checked by analytical reverse
phase high pressure liquid chromatography, thin layer
chromatography and photon counting spectrofluorometry.
EXAMPLE 5
Preparation of a Polypeptide Derivative of Rhodamine 110 Employing
the HOBT-BOP Procedure
A 4-fold excess of the FMOC polypeptide and a 4-fold excess of HOBT
and BOP are placed into a round bottom flask containing a 0.6
millimolar solution of N-methylmorpholine in dimethylformamide and
stirred for 10-15 minutes. To this solution is added dropwise a
solution of the monopeptide rhodamine 110 dissolved in a minimum
amount of a 0.6 millimolar solution of N-methylmorpholine in
dimethylformamide. This addition requires 5-10 minutes, and the
reaction is stirred at room temperature for four hours. The
reaction solution is concentrated under reduced pressure to an oil.
This oil is dissolved in methylene chloride and the crude product
is purified by normal phase HPLC. The eluate containing the desired
product is collected and concentrated under reduced pressure
affording a crystalline material. The purity and identify of this
material are checked by analytical reverse phase HPLC and thin
layer chromatography. The FMOC blocking is removed by dissolving
the solid in a 5% piperidine-dimethylformamide solution and stirred
at room temperature for one hour. The solution is concentrated
under reduced pressure, and the resulting solid is triturated
several times with pentane to remove the FMOC polymer. The
remaining solid is dissolved in a minimum of methanol and a 5-fold
excess of trifluoroacetic acid is added. The solution is
concentrated under reduced pressure and the resulting solid is
centrifuged with cold diethyl ether until the ether triturate has a
pH=7. If the polypeptide is polar then the remaining protective
group(s) is removed by treating with a 30 to 50% trifluoroacetic
acid solution in methylene chloride for four hours at room
temperature. The solution is concentrated under reduced pressure to
dryness, and the resulting solid is centrifuged with cold diethyl
ether until the ether triturate has a pH=7. A final purification of
this trifluoroacetic acid substrate is effected with reverse phase
HPLC. The eluate containing the product is concentrated under
reduced pressure and the aqueous solution is lyophilized. The
product's purity and identify are checked by analytical reverse
phase HPLC, thin layer chromatography and photon counting
spectrofluorometry.
EXAMPLE 6
Preparation of p-Aminobenzoic Acid Derivative of Rhodamine 110
A molar quantity of p-Aminobenzoic Acid is placed into a round
bottom flask containing a small quantity of dioxane and stirred
until a complete solution occurs. A 10% molar excess solution of
sodium carbonate, dissolved in water, is added. To this
well-stirred solution is added dropwise a molar solution of
9-fluorenylmethyloxycarbonylchloride dissolved in a minimum of
dioxane. This addition requires 10 to 15 minutes and the reaction
solution is allowed to stir an additional four (4) hours. The
reaction is diluted with water and extracted (3) times with diethyl
ether. The aqueous layer is cooled in an ice water bath and the pH
is adjusted to two (2) with a 10% solution of hydrochloric acid.
The resulting colorless precipitate is filtered and recrystallized
from an acetone solution. A TLC of the colorless, crystalline
product showed only one (1) quenched spot and obtained in a yield
of 68%.
A 6-fold excess of the FMOC-p-amino acid is placed into a round
bottom flask containing a 50:50 pyridine-dimethylformamide solution
(V:V) and stirred until a complete solution occurs. To this stirred
solution is added a 12-fold excess of EDAC and the admixture is
stirred for 30 minutes. A solution of rhodamine 110, dissolved in a
minimum of a 50:50 pyridine-dimethylformamide (V:V) is added
dropwise to the reaction solution. This addition requires 15-20
minutes, and the reaction solution is concentrated under reduced
pressure to an oil and dissolved in a small amount of chloroform.
The crude product is purified by normal phase HPLC and the product
is eluted in a 5% methanol-chloroform solution. This eluate is
concentrated under reduced pressure, and the resulting colorless
solid dried in vacuo affording a 60% yield of the product. This
material is treated with a 5% solution of piperidine dissolved in
dimethylformamide. The resulting solution is stirred at room
temperature for one (1) hour and concentrated under reduced
pressure. The resulting solid is triturated several times with
pentane, dissolved in a minimum of methanol and a 5-fold excess of
trifluoroacetic acid is added. The solution is concentrated to
dryness and the resulting solid is centrifuged with cold diethyl
ether until the ether triturate has a pH of seven (7). The
trifluoroacetate salt is dried in vacuo overnight affording a yield
of 74.5%. The product's purity and identify are checked by
analytical reverse phase high pressure liquid chromatography, thin
layer chromatography and photon counting spectrofluorometry.
EXAMPLE 7
Preparation of Tetraacetyl-.alpha.-D-Glucopyranosyl Derivative of
Rhodamine 110 and Tetrabenzoyl-.alpha.-D-Glucopyranosyl Derivative
of Rhodamine 110
A 10-fold excess of the respective protected tetraacetyl (or
tetrabenzoyl) .alpha.-D-glucopyranosyl bromides is placed into a
round bottom flask containing a 50:50 pyridine-dimethylformamide
solution (V:V) and warmed and stirred until a complete solution
occurs. To this stirred solution is added a 12-fold excess of EDAC
and the admixture is stirred for 30 minutes. A solution of
rhodamine 110, dissolved in a minimum of a 50:50
pyridine-dimethylformamide (V:V) is added dropwise to the reaction
solution. This addition requires 15 to 20 minutes. The reaction
solution is allowed to stir for 24 hours and concentrated under
reduced pressure to an oil. The crude product is dissolved in
chloroform and purified by normal phase HPLC. The
tetraacetyl-.alpha.-D-glucopyranosyl derivative is eluted in a 1%
methanol-chloroform solution. The
tetrabenzoyl-.alpha.-D-glucopyranosyl derivative is eluted in a 3%
methanol-chloroform solution. The product is isolated by
concentrating the respective eluates under reduced pressure and
drying in vacuo overnight. The yield of the
tetraacetyl-.alpha.-D-glucopyranosyl rhodamine 110 is 30% and the
yield of the tetrabenzoyl-.alpha.-D-glucopyranosyl rhodamine 110 is
almost quantitative (100%). The product's purity and identity are
checked by analytical reverse phase high pressure liquid
chromatography, thin layer chromatography and photon counting
spectrofluorometry.
EXAMPLE 8
Preparation of N-Butyl Ester Derivative of Fluorescein
A 3.4 fold excess of n-butyrylanhydride is placed into a round
bottom flask containing a minimum amount of tetrahydrofuran and
stirred for several minutes. A one molar equivalent of fluorescein
is added, followed by 10 mL of triethylamine and solution occurs.
The reaction solution is stirred an additional 30 minutes and then
concentrated under reduced pressure to an oil. The oil is dissolved
in a minimum of chloroform and the crude product is purified by
normal phase HPLC. The product is eluted with a 0.5%
methanol-chloroform solution and the eluate is concentrated under
reduced pressure affording a yield of a colorless solid of 63%. The
product's purity and identity are checked by analytical reverse
phase high pressure liquid chromatography, thin layer
chromatography and photon counting spectrofluorometry.
EXAMPLE 9
Preparation of Chloroacetyl Ester Derivative of Fluorescein
A 10-fold excess of chloroacetic anhydride is placed into a round
bottom flask containing a minimum amount of tetrahydrofuran and
stirred for several minutes. A solution of fluorescein, dissolved
in a minimum amount of tetrahydrofuran and a 2-fold excess of
triethylamine, is added dropwise to the reaction mixture. This
addition required 10 to 15 minutes and the reaction solution is
allowed to stir overnight. The solution is concentrated under
reduced pressure to an oil. This oil is dissolved in a minimum
amount of methylene chloride and the crude product is purified by
normal phase HPLC. The desired product is eluted in a 100%
methylene chloride solution, and this eluate concentrated under
reduced pressure. This solid is dried in vacuo for 16 hours
affording a quantitative yield (100%) of the desired product. The
product's purity and identity are checked by analytical reverse
phase high pressure liquid chromatography, thin layer
chromatography and photon counting spectrofluorometry.
EXAMPLE 10
Preparation of n-Palmityl Ester Derivative of Fluorescein
A 2.5-fold excess of palmitic acid is placed into a round bottom
flask containing a minimum amount of tetrahydrofuran and stirred
for several minutes. To this solution is added a 3-fold excess of
EDAC and the mixture stirred for 30 minutes. A solution of
fluorescein dissolved in a minimum amount of tetrahydrofuran is
added dropwise to the reaction mixture. This addition required 15
to 20 minutes and the reaction mixture is allowed to stir
overnight. The reaction mixture is concentrated under reduced
pressure to an oil. This oil is dissolved in chloroform and
extracted three (3) times with a 5% aqueous sodium bicarbonate
solution. The organic layer is dried over magnesium sulfate,
filtered and concentrated to dryness. This crude product is
purified by normal phase KPLC and the product is eluted with 100%
chloroform. This eluate is concentrated under reduced pressure and
the resulting solid is dried in vacuo affording 400 mg (9% yield).
The product's purity and identity are checked by analytical reverse
phase high pressure liquid chromatography, thin layer
chromatography and photon counting spectrofluorometry.
An elemental analysis for carbon and hydrogen, by Galbraith
Laboratories, Inc. of Knoxville, Tenn., gave the following results:
Formula: C.sub.52 H.sub.72 O.sub.7. 1/2H.sub.2 O MW=817,075
______________________________________ Theoretical Found
______________________________________ C = 76.34 C = 76.36; 76.51 H
= 8.99 H = 9.01; 9.05 ______________________________________
EXAMPLE 11
Preparation of Diphenylphosphate Derivative of Rhodamine 110
A 6.6-fold excess of diphenylchlorophosphate is placed into a round
bottom flask containing a very small amount of pyridine and stirred
several minutes in an ice-water bath. To this well-stirred, cold
solution is added rhodamine 110 and a white precipitate is formed
immediately. The stirring is continued for an hour and the reaction
mixture is placed in the refrigerator for 48 hours. The reaction
mixture is treated with water and extracted twice with chloroform.
The combined chloroform extracts are dried over magnesium sulfate,
filtered and concentrated under reduced pressure. The crude product
is purified by normal phase HPLC. The desired product is eluted in
a 1% methanol-chloroform solution, and this eluate is concentrated
under reduced pressure to an oil. This oil is dissolved in ammonia,
and the resulting aqueous solution is lyophilized affording the
ammonium salt of the product in a 66% yield. The product's purity
and identity are checked by analytical reverse phase high pressure
liquid chromatography, thin layer chromatography and photon
counting spectrofluorometry.
EXAMPLE 12
Preparation of Diphenylphosphate Derivative of Fluorescein
A 6.6-fold excess of diphenylchlorophosphate is placed into a round
bottom flask containing a very small amount of pyridine and stirred
several minutes in an ice-water bath. To this well-stirred, cold
solution is added fluorescein and a white precipitate is formed
immediately. Stirring is continued for one (1) hour and the
reaction mixture placed in the refrigerator for 48 hours. The
reaction mixture is treated with water and extracted twice with
chloroform. The combined chloroform extracts are dried over
magnesium sulfate, filtered and concentrated to dryness under
reduced pressure. The resulting solid is dissolved in a minimum
amount of chloroform and the crude product is purified by normal
phase HPLC. The product is eluted in a 1% methanol-chloroform
solution and this solution concentrated under reduced pressure. The
colorless solid is treated with ammonia and lyophilized, affording
the ammonium salt in a yield of 95%. The product's purity and
identity are checked by analytical reverse phase high pressure
liquid chromatography, thin layer chromatography and photon
counting spectrofluorometry.
EXAMPLE 13
Preparation of Trifluoroacetyl Ester Derivative of
4'(5')Carboxyfluorescein
A 10-fold excess of trifluoroacetic anhydride is placed into a
round bottom flask containing a minimum amount of tetrahydrofuran
and stirred several minutes. A 30% pyridine-tetrahydrofuran
solution, containing the 4'(5')carboxyfluorescein, is added
dropwise over 10 to 15 minutes. The solution is allowed to stir
overnight and concentrated under reduced pressure. The resulting
oil is dissolved in chloroform, extracted three times with water
and the organic layer dried over magnesium sulfate. This is
filtered, concentrated to a small volume under reduced pressure and
the crude product purified by normal phase HPLC. The product is
eluted in a 4% methanol-chloroform solution. This is concentrated
under reduced pressure and resulting solid dried in vacuo for 15
hours affording an 83% yield. The product's purity and identity are
checked by analytical reverse phase high pressure liquid
chromatography, thin layer chromatography and photon counting
spectrofluorometry.
EXAMPLE 14
Preparation of Diphenylphosphate Ester Derivative of
4'(5')Carboxyfluorescein
A 15-fold excess of chlorodiphenylphosphate is added to a solution
of 4'(5')carboxyfluorescein, dissolved in 8 mL of pyridine over a
period of 10 to 15 minutes. The reddish-colored solution turns a
light yellow and a precipitate is formed. Stirring is continued for
two hours and the mixture is allowed to cool in the refrigerator
overnight. To this mixture is added 100 mL of water and the mixture
extracted three times with chloroform. The combined chloroform
extracts are dried over magnesium sulfate, filtered and
concentrated under reduced pressure to an oil. This oil is
dissolved in a minimum amount of methylene chloride, and the crude
product is purified by normal phase HPLC. The desired product is
eluted in a 1% methanol-chloroform solution, and this eluate is
concentrated under reduced pressure. The resulting solid is dried
in vacuo for 16 hours affording a yield of 95%. The product's
purity and identity are checked by analytical reverse phase high
pressure liquid chromatography, thin layer chromatography and
photon counting spectrofluorometry.
EXAMPLE 15
Preparation of H-L-Leucine Trifluoroacetate Salt Derivative of
Rhodol
A 10-fold excess of benzyloxycarbonyl-L-leucine is placed into a
round bottom flask containing a 50:50 pyridine-dimethylformamide
solution (V:V) and stirred until a complete solution occurs. To
this stirred solution is added a 12-fold excess of EDAC and the
admixture is stirred for 30 minutes. A solution of rhodol
hydrochloride, dissolved in a minimum of a
pyridine-dimethylformamide solution (V:V), is added dropwise to the
reaction solution. This addition required 10 to 15 minutes and the
reaction is allowed to stir at room temperature overnight. The
solution is concentrated under reduced pressure to an oil. This oil
is dissolved into chloroform and extracted three (3) times with
water and organic layer dried over magnesium sulfate. This is
filtered, concentrated to a-very small volume under reduced
pressure and purified by normal phase HPLC. The product is eluted
in a 2% methanol-chloroform solution. This eluate is concentrated
under reduced pressure and the resulting colorless, crystalline
solid, dried in vacuo, affords a 33.4% yield of the product and the
product's purity and identity are checked by analytical reverse
phase high pressure liquid chromatography and thin layer
chromatography. This material is dissolved into a small volume of
isopropyl alcohol and catalytically reduced with a small amount of
10% palladium on carbon as the catalyst in a Paar shaker apparatus
for 16 hours. The alcohol solution is carefully filtered and a
2-fold excess of trifluoroacetic acid is added. This solution is
concentrated to dryness under reduced pressure and the resulting
solid is centrifuged with cold diethyl ether until the ether
triturate has a pH of 7. The colorless trifluoroacetate salt is
dried in vacuo overnight, affording a 92.48% yield. The product's
purity and identity are checked by analytical reverse phase high
pressure liquid chromatography, thin layer chromatography and
photon counting spectrofluorometry.
EXAMPLE 16
Preparation of (H-LEU-GLY).sub.2 Rhodamine 110 Acetate and Tartrate
Salts
A 10-fold excess of FMOC glycine is placed into a round bottom
flask containing a 50:50 pyridine-dimethylformamide solution (v:v)
and stirred until a complete solution occurs. To this stirred
solution is added a 12-fold excess of EDAC and the admixture is
stirred for 30 minutes. A solution of rhodamine 110 is dissolved in
a minimum of a 50:50 pyridine-dimethylformamide solution (v:v) and
is added dropwise to the reaction solution. This addition requires
15-20 minutes, and the reaction is allowed to stir at room
temperature overnight. The solution is concentrated under reduced
pressure to an oil. This oil is dissolved in a small amount of
methylene chloride, and the product is purified by normal phase
EPLC. The product is eluted from the column in a 1%
methanol-chloroform solution. This eluate is concentrated under
reduced pressure, and the resulting solid dried in vacuo affording
an 85% yield of the product. The purity and identity are checked by
analytical reverse phase high pressure liquid chromatography and
thin layer chromatography. This material is treated with a 5%
solution of piperidine dissolved in dimethylformamide. The
resulting solution is stirred at room temperature for one (1) hour
and concentrated under reduced pressure. The resulting solid is
triturated several times with pentane and product dried in vacuo. A
TLC of this material showed only one quenched spot which is
positive to concentrated hydrochloric acid.
A four (4) fold excess of FMOC-L-leucine is placed into a round
bottom flask containing a 50:50 pyridine-dimethylformamide solution
(v:v) and stirred until a complete solution occurs. To this stirred
solution is added an eight (8) fold excess of EDAC and the
admixture is stirred for 30 minutes. A solution of (H-GLY).sub.2
rhodamine 110 (from above) dissolved in a minimum of a 50:50
pyridine-dimethylformamide solution (v:v) is added dropwise to the
reaction solution. This addition requires 15-20 minutes, and the
reaction is allowed to stir at room temperature for six (6) hours.
The solution is concentrated under reduced pressure to an oil. This
oil is dissolved in chloroform, and the product is purified by
normal phase high pressure liquid chromatography. The product is
eluted from the column in a 2% methanol-chloroform solution. This
eluate is concentrated under reduced pressure, and the resulting
solid dried in vacuo affording a 74% yield. The purity and identity
are checked by analytical reverse phase high pressure liquid
chromatography and thin layer chromatography. This material is
treated with a 5% solution of piperidine dissolved in
dimethylformamide. The resulting solution is stirred at room
temperature for one (1) hour and concentrated under reduced
pressure. The solid is triturated several times with pentane and
dried in vacuo. One half of this material is dissolved in a small
amount of methanol and a 10% excess of acetic acid is added. Ether
is added to this solution and cooled in an ice-water bath. The
resulting colorless solid is filtered, washed with ether and
centrifuged with ether until the pH=7. The crystalline salt is
dried in vacuo according a 61% yield.
The remaining one-half of the material (from above) is dissolved in
a small amount of methanol and a 10% excess of L-tartaric acid
dissolved in a very small amount of methanol is added. This
solution is cooled in an ice-water bath and ether is added. The
resulting crystalline material is filtered, washed with ether and
centrifuged with ether until the pH=7. The resulting, colorless
salt is dried in vacuo affording a 42% yield. The free fluorescence
and identity of the acetate and tartrate salts of these rhodamine
110 substrates are checked by analytical reverse phase high
pressure liquid chromatography, thin layer chromatography and
photon counting spectrofluorometry.
The acetate and tartrate salts thus prepared have the following
characteristics, respectively: native free fluorescence, 63,000 and
61,000 photons; autohydrolysis rate when measured at 37.degree. C.
using a 1 cm path length, -5.56 and -3.8 change in photons per
second; and enzymatic reaction rate of cathepsin B at 37.degree.
C., +128 and +138 change in photons per second. The purity and
stability of acetate and tartrate salts of (LeuGly).sub.2 rhodamine
110 as demonstrated by assessment of autohydrolysis, background
fluorescence and enzymatic activity after storage at 4.degree. C.
are illustrated in FIGS. 12A-12D as follows:
FIG. 12A (background fluorescence, Leu-Gly.cndot.acetate);
FIG. 12B (autohydrolysis and enzyme rate,
Leu-Gly.cndot.acetate);
FIG. 12C (background fluorescence, Leu-Gly.cndot.tartrate); and
FIG. 12D (autohydrolysis and enzyme rate,
Leu-Gly.cndot.tartrate).
EXAMPLE 17
Preparation of the Free Amine of (Lys-Ala).sub.2 Rhodamine 110
A 10-fold excess of the FMOC L-lysine .epsilon.BOC amino acid is
placed into a round bottom flask containing a 50:50
pyridine-dimethylformamide solution (V:V) and stirred several
minutes. To this well-stirred solution is added a 20-fold excess of
EDAC and the admixture is stirred an additional 30 minutes. A
solution of (Ala).sub.2 rhodamine 110 dissolved in a minimum of
50:50 pyridine-dimethylformamide solution (V:V) is added dropwise
over a period of 15 to 20 minutes. The reaction is stirred at room
temperature for 16 hours and then concentrated to an oil under
reduced pressure. This oil is dissolved in a minimum of an organic
solvent and the crude product is purified by normal phase HPLC. The
eluate containing the desired product is collected and concentrated
under reduced pressure affording a crystalline material. A TLC of
this material is run to check for purity and identity. The BOC
protecting group is removed by dissolving the solid into a 50%
solution of trifluoroacetic acid in methylene chloride. The
reaction is stirred at room temperature for one hour, and the
purity of the reaction product is checked by thin layer
chromatography. The TLC did not show any of the BOC group. The acid
solution is concentrated under reduced pressure to dryness. Several
washes with fresh methylene chloride and reconcentrations under
reduced pressure are performed to generate a crystalline solid. The
FMOC blocking group is removed by dissolving the solid in a 5%
piperidine-dimethylformamide solution and stirred at room
temperature for one hour. The solution is concentrated to dryness
under reduced pressure. The resulting solid is triturated several
times with pentane to remove the FMOC polymer and the product is
dried in vacuo to constant weight affording a yield of 98.62%. The
purity of this material is checked by reverse phase HPLC,
thin-layer chromatography and photon counting spectrofluorometry.
The stability and purity of the product is further determined by
monitoring the autohydrolysis, background fluorescence and
enzymatic activity with the product as a substrate after storage of
the product at 4.degree. C. FIGS. 13A and 13B illustrate the
stability of the free amine of (Lys-Ala).sub.2 rhodamine 110 which
was prepared by the procedure described in this Example. FIG. 13A
shows background fluorescence and FIG. 13B shows autohydrolysis and
enzyme rate.
EXAMPLE 18
Use of Different Salts to Enhance Specificity
The use of salts to identify cellular enzymes is very important. pH
optimas are different demonstrating different enzymes or
isoenzymes. Different salts from within the same pH range may give
different reactivities. Z-groups, which are not salts but covalent
organic compounds, show relatively little activity and no pH
optima. See FIGS. 2A-2D, using cathepsin B as a target enzyme.
EXAMPLE 19
Use of Inhibitors in the Reagent Formula
Use of inhibitors of the targeted enzyme has been shown to prove
substrate specificity. More specifically, when an inhibitor
eliminated the targeted enzyme signal, it was reasoned that the
targeted enzyme activity was measured without the inhibitor. The
disclosed enzyme assay contemplates the use of interfering reaction
inhibitors to increase and maintain specificity.
To improve a Cathepsin D response, inhibitors to aminopeptidase and
Cathepsin B are added to the substrate most specific for Cathepsin
D. Conversely, adding a Cathepsin D inhibitor to an assay for
Cathepsin D requires measurement before and after inhibitor
addition thus requiring two (2) measurements per assay. The
opposite approach only requires one measurement. See FIGS.
3A-3D.
EXAMPLE 20
Immune Competence
The cell's ability to fight off an invader lies within its genetics
and therefore cell type. The "readiness" however of any genetically
capable group of cells to defend is different. A measure of this
"readiness" is manifest in the available proteolytic enzymes
contained within vacuoles or on the surface of the cell. The assay
compound hydrolysis rate increases with increased mass of enzymes
giving a picture of immune competence both in number of cells and
activity level. FIG. 4A shows cell size (fs), granularity (ss) and
amino peptidase activity (log fluorescence at 525 nm v. time) using
Leu rhodamine 110-TFA as a substrate in normal Ficoll prepared
lymphocytes. FIG. 4B shows the same data for acute lympholytic
Ficoll prepared lymphocytes. The cells tested in FIG. 4B have lost
their enzymatic activity. Images were generated using Universal
Imaging.
EXAMPLE 21
Leukemia
A panel of assay compounds are assembled consisting of
pro-aminopeptidase; aminopeptidase M (Pro, Lys, Gly, Ala, Leu),
Cathepsin D (Gly-Leu, Thr-Pro), Cathepsin B (Gln-Ser, Leu-Gly,
Val-Ser, Val-Lys), Cathepsin C (Ala-Gly) and dipeptidyl peptidase
II (Lys-Ala, Gly-Pro, Ala-Ala). Values for these assay compounds
outside the normal range are considered diagnostic for leukemia. In
addition, the ratios of these enzyme readings to one another
provide information on further classifying the leukemia into
myelogenous or lymphocytic and monitoring the course of the
disease. Values may be both higher or lower than the normal range.
FIG. 5A shows results obtained when normal leukocytes are tested
with various rhodamine 110-monopeptide and rhodamine 110-dipeptide
compounds. All compounds except (Lys-Ala).sub.2 rhodamine 110 are
TFA salts. (Lys-Ala).sub.2 rhodamine 110 was a free amine
derivative. FIG. 5B shows results obtained when leukemia cells are
tested with various rhodamine 110-monopeptide compounds.
EXAMPLE 22
Sepsis
A panel of assay compounds are assembled consisting of
aminopeptidase (Leu, Pro, Lys, Gly, Ala), dipeptidyl peptidase II
(Gly-Pro, Lys-ala, Ala-Ala), Cathepsin C (Ala-Gly) and Cathepsin B
(Leu-Gly, Val-Lys, Val-Ser and Gln-Ser) and cathepsin D (Gly-Leu
and Thr-Pro). Values for these substrates outside the normal range
are considered diagnostic for sepsis. FIG. 6A shows results
obtained when cells from a patient that had been shot by a gun and
who was experiencing sepsis were treated with various rhodamine
110-monopeptide and dipeptide compounds. FIG. 6B shows results
obtained when umbilical cord blood cells from a newborn infant were
treated with various rhodamine 110-monopeptide and dipeptide
compounds. All compounds except (Lys-Ala).sub.2 rhodamine 110 are
TFA salts. (Lys-Ala).sub.2 rhodamine 110 was a free amine
derivative.
EXAMPLE 23
TB Infection
A panel of assay compounds are assembled consisting of
Ala-aminopeptidase and Lys-aminopeptidase, Dipeptidyl peptidase IV
(Ata-Ala).sub.2 rhodamine 110 and Cathepsin D to indicate possible
TB infection in AIDS related cases.
A panel of enzymatic substrates is performed consisting of
Ala-aminopeptidase and Lys-aminopeptidase, Dipeptidyl peptidase IV
(Ala-Ala).sub.2 rhodamine 110 and Cathepsin D to indicate possible
TB infection in AIDS related cases. The results are reported in
Table 4 below:
TABLE 4 ______________________________________ NORMALS HIV +
PATIENTS MEAN MEAN DELTA DELTA P SUBSTRATE FL SD N FL SD N 2 TAIL
______________________________________ GLN-SER**** 11.9 4.4 14 13.7
7.2 13 N.S. GLN-SER* 6.1 2.6 14 12.7 12.9 17 <0.060 VAL-SER****
24.6 5.3 14 32.5 14.7 7 N.S. LYS-ALA.sup.1 112.6 43.5 12 121.5 62.9
7 N.S. LYS-ALA.sup.2 7.6 1.7 12 7.9 3.7 13 N.S. THR-PRO**** 295.0
140.9 7 204.4 106.1 6 N.S. ALA-GLY**** 48.0 44.1 11 39.9 43.7 6
N.S. ALA-GLY* 31.6 27.0 13 29.7 24.8 13 N.S. THR-PRO**** 87.2 95.8
13 62.9 99.2 13 N.S. THR-PRO* 17.3 18.5 13 10.2 8.7 17 N.S.
GLY-PRO**** 46.7 42.8 17 18.4 9.2 13 <0.020 GLY-PRO* 50.9 45.9
17 21.4 15.5 19 <0.025 ALA-ALA**** 158.4 47.5 9 110.1 31.6 16
<0.020 ALA-ALA* 25.6 7.6 13 26.3 10.4 13 N.S. GLY-LEU**** 9.5
4.7 17 16.3 11.1 17 <0.030 LEU-GLY** 143.0 132.7 13 104.1 98.4
13 N.S. LEU-GLY*** 162.4 146.9 13 111.9 99.8 13 N.S. VAL-LYS****
22.7 16.1 15 17.7 10.4 13 N.S. GLYCINE**** 101.1 101.2 11 62.4 73.3
13 N.S. ALANINE**** 132.0 140.4 13 59.0 31.3 17 <0.095
LYSINE**** 10.5 3.7 12 7.4 3.8 17 <0.040 PROLINE**** 11.4 4.3 10
11.0 5.0 13 N.S. LEUCINE**** 259.6 99.6 8 130.5 99.5 9 <0.020
______________________________________ *BESTATIN + TFA RHODAMINE
110 **TARTRATE ***ACETATE ****TFARHODAMINE 110 .sup.1 Rho 110free
amine .sup.2 Rho 110free amine + bestatin
EXAMPLE 24
Metastatic Potential in Solid Tumors
A panel of assay compounds are assembled consisting of Cathepsin B
markers (Gln-Ser, Val-Ser), Cathepsin C (Thr-Pro), Dipeptidyl
peptidase IV (Ala-Ala) and Leu-aminopeptidase to predict metastatic
potential in solid tumors. The results obtained when breast tumor
cells (and one normal breast control cell sample) are treated with
TFA salts of various rhodamine 110-peptide compounds are shown in
FIG. 7.
EXAMPLE 25
Monitoring Drug Treatment
An assay compound can be used to monitor drug treatment. Enzymatic
activity according to drug target, i.e., protein synthesis, can
diminish over time and increase dramatically depending on dose of
drug. The results obtained when Raji cells which had been exposed
to various concentrations of cyclophosphamide for 48 hours are
treated with Leu-rhodamine 110 for 1 minute are shown in FIG. 8A.
The results obtained when Raji cells, which had been exposed to
various concentrations of vincristine for 48 hours, are treated
with a TFA salt of Leu-rhodamine 110 for 1 minute and
aminopeptidase activity is measured are shown in FIG. 8B.
EXAMPLE 26
Macrophage Activation
FIG. 9 illustrates the use of assays to provide an indication of
macrophage activation. Using a mouse model, various eypes of cells
used to study metastatic versus non-metastatic breast tumors, were
treated with Leu-rhodamine 110 substrate and amino peptidase
activity was measured. The results obtained are shown in FIG.
9.
EXAMPLE 27
Red Blood Cell Adenosine Deaminase (ADA) and Relationship To
Hereditary Non-Spherocytic Hemolytic Anemia (HNSHA) Disease
Hereditary deficiencies of glycolytic enzymes or related pathways
in the erythrocyte are characterized by the disease hemolytic
anemia. Hereditary Non-Spherocytic Hemolytic Anemia is
distinguished from Hereditary Spherocytosis by the fact that red
blood cells are morphologically normal and manifest a normal
osmotic fragility. Only in the case of pyrimidine 5' nucleotidase
deficiency is the erythrocyte morphology changed to a basophilic
stippling.
Deficiencies of ADA are well-known causes of immunodeficiency. In
cases where ADA is greatly increased to levels as high as 100 times
normal but other tissues have normal levels in the same individual,
the clinical disease is HNSHA. The high ADA depletes the
erythrocytes of vital adenine nucleotides, impairing their
metabolism. The residual enzyme structure is normal and the gene is
normal but attaching the promoter to a reporter gene produced
increased levels of enzyme.
The structure of Adenosine is: ##STR2##
The enzyme adenosine deaminase removes the NH.sub.2 and replaces it
with a hydroxyl: ##STR3##
The assay compound using rhodamine 110 is then: ##STR4##
Hydrolysis by ADA leaves 2 Inosine and 1 rhodamine 110:
##STR5##
To assay vital cells for ADA activity, a blood sample (containing
platelets, erythrocytes and leukocytes) is washed to remove plasma,
debris, dead cells and extra cellular enzymes. The sample is
incubated at 37.degree. C. in the wash media.
A media is prepared for the assay reagent using Hanks balanced
salts at pH 7.0. The aqueous buffer media is adjusted to isosmotic
conditions. The ionic strength is adjusted to 0.1 to 0.3.mu. by
additions of salts. Appropriate cofactors including divalent
cations such as Ca.sup.2+, Mg.sup.2+ and Ba.sup.2+ are added for
ADA to maximize the hydrolysis rate. The assay compound is added at
excess for the quantities of enzyme analyzed. (A time-course
activity assay is used to determine correct fluorescence intensity
data collection, usually between 10 seconds and 10 minutes, as well
as, appropriate assay compound concentration.)
The washed, pre-incubated blood sample is added to the media,
incubated at 37.degree. C. and fluorescent intensity is measured at
the predetermined time on erythrocytes. The fluorescence found on
platelets and leukocytes are disregarded. Separation of cell types
is aided by size discrimination.
Comparison of "normal" erythrocytic ADA activity to those in HNSHA
disease state demonstrates a 100-fold increase in ADA activity.
The structure of pyrimidine 5'nucleotide is: ##STR6##
The enzyme pyrimidine 5'nucleotidase removes the phosphate group
from the compound: ##STR7##
The assay reagent using fluorescein is then: ##STR8##
To assay vital cells for pyrimidine-5'-nucleotidase, a blood sample
containing platelets, erythrocytes, leukocytes and plasma is washed
to remove plasma, debris, dead cells and extracellular enzymes. The
sample is pre-incubated at 37.degree. C. in the wash media.
A media is prepared for the assay reagent using glycine-sodium
hydroxide buffer at pH 8.5. The aqueous buffer media is adjusted to
isosmotic conditions. The ionic strength is adjusted to 0.1 to
0.3.mu. by addition of salts. Appropriate cofactors of calcium
chloride and magnesium sulfate are added for pyrimidine
5'nucleotidase to maximize the hydrolysis rate. The assay compound
is added at excess for the quantities of enzyme to be analyzed.
Other Michaelis-Menten parameters are determined to provide correct
data collection window.
The washed pre-incubated blood sample is added to the media,
incubated at 37.degree. C. and fluorescent intensity is measured on
erythrocytes. Erythrocytes are identified visually under a
microscope using morphological indicators. Erythrocytes are
identified using a flow cytometer by size and granularity
discrimination or 2-color assay monoclonal antibody for
erythrocytes.
Comparison of "normal" erythrocytes pyrimidine 5'nucleotidase to
those in HNSHA shows a deficiency of enzyme in HNSHA.
EXAMPLE 28
Proinsulin or Pre-proinsulin Inside Cell
Insulin is synthesized as a single chain
polypeptide--pre-proinsulin. The signal sequence "pre" becomes
cleaved during synthesis on the rough endoplasmic reticulum, and no
mutations are known that cause disturbances of removing signal
sequences, because such mutations probably would be lethal.
Proinsulin is characterized by the presence of a C-peptide that
joins the two A and B chains of the mature insulin molecule.
Mutations occur mostly at the two critical junctions where the
C-peptide is attached to the A and B insulin chains by two pairs of
basic amino acids. Such defects have been recognized in families
with hyperinsulinemia. The defect is inherited in an autosomal
dominant pattern, and probably involves the loss of one of the
basic amino acid residues that makes it impossible to cleave the
proinsulin molecule at the mutation site, which results in the
presence of a two-chained intermediate of proinsulin molecules
secreted into the blood plasma.
Clinically occurring glucose intolerance with abnormally high
ratios of proinsulin-like material to insulin (9 to 10 as compared
with normal values of approximately 0.25) are due to a loss of Arg
65 and loss of the protective activity causing acetylation of Lys
64 which make it impossible to cleave the C-peptide from the A
chain of insulin. It is supposed that substitution of Arg 65 of the
proinsulin molecule results in failure of cellular enzymes to
cleave correctly the C-peptide from the A chain of insulin. Use of
a specific substrate, designed for this cleavage site, can be
diagnostic for the defect, as well as prognostic for genetic
treatment.
EXAMPLE 29
Hairy Cell Leukemia
Acid-tartaric buffer in phosphate substrate is used to confirm
hairy cell leukemia. "Tartaric Resistant Acid Phosphatase" is a
cellular component found in hairy cell leukemia. Design of a buffer
system, using tartrate in the buffer and a phosphate substrate,
confirms presence of this disease with a positive result.
EXAMPLE 30
Cathepsin B
Cathepsin B substrates Gln-Ser, Val-Ser, Leu-Gly, Val-Lys specific
for isoenzymes of Cathepsin B. The use of isoelectric focusing for
Cathepsin B enzyme and nitrocellulose transfer of these isoenzymes,
similar to Western blot, provides a solid support to test substrate
activity. Examination of substrate activity, based on design of the
dye molecule, determines isoenzyme specificity based on
structure.
To prepare the reagent for measuring cathepsin B activity, an assay
compound such as Val-Lys-rhodamine 110-TFA is dissolved in 100%
DMSO at a stock concentration of 1.6 mM. The stock solution is then
diluted 20-fold [with 10 mM MES buffer at pH 6.0] to give a reagent
concentration of 0.08 mM. To the 0.08 mM solution the following
reagents are added. 0.5 mM Bestatin as an aminopeptidase inhibitor,
1.0 mM dithiothretol as activator, 1.0 mM CaCl.sub.2 and 1.0
MgCl.sub.2 as cofactors and 247 mM Mannitol as a bulking reagent
for lyophilization. The complete reagent mixture is lyophilized
during which process the DMSO is effectively removed and the
lyophilized mixture is reconstituted using endotoxin-free deionized
water.
EXAMPLE 31
Response to Modulators
The use of cellular response modifiers, i.e., PMA (phorbol
myristate acetate), interleukins and interferons in the
pre-incubation step provides information on cellular response. If
the cell function is normal the response to the modulator will, for
selected enzyme substrates, be in defined ranges. If abnormal, the
measured response will be higher or lower.
To make the reagent, the assay compound dichlorofluoroscein
diacetate is dissolved in 100% DMSO at 6.0 mM as a stock solution.
The stock solution is diluted 100-fold with 10 mM MES at pH 6.0 to
give a reagent concentration of 0.06 mM. 0.032 mM PMA is added as a
cell-activator and 247 mM Mannitol is added as a bulking agent for
lyophilization. The reagent mixture is lyophilized which
effectively removes the DMSO and reconstituted with endotoxin-free
deionized water.
EXAMPLE 32
Use in Conjunction with Genetic Analysis Techniques
The methods to determine the activity of an enzyme using the assays
described above are also useful when used in independent
combination with genetic analysis techniques including, polymerase
chain reaction (PCR), transcription mediated amplification (TMA),
ligase chain reaction (LCR) and fluorescent in situ hybridization
(FISH). The results obtained using these genetic analysis
techniques can be used both for confirmation of diagnostic
conclusions based on measurements of enzymatic activities in cells,
as determined with the assays previously described herein, and for
the differentiation of purely functional pathologies and functional
pathologies having an underlying genetic cause.
EXAMPLE 33
Statistical Analysis and Diagnosis of Normal and Diseased States
based on Cellular Enzymatic Activity
A normal in house adult donor pool was drawn, recording sex and
determining that no known disease state was present at the time of
sample draw. Criteria for rejecting samples included patients under
medication, patients with infection or inflammation of any type,
colds and flu as well as any known medical illness such as cancer,
heart ailment and high blood pressure. A donor list of 75 patients
has been developed and a mean and 2 sd range established. A
subgroup of "super normals" was developed from this list by
examining the 75 patients to determine which group falls closest to
the mean for all enzymes in all cell types tested. These patients
were used as the "wellness index" or best group of "normalcy"
patients. If a study required the use of children or newborns then
this same format was used to develop a data base for these age
groups. It was noted that the normal range was different in the
different age groups. These groups were also screened for criteria,
for example in the newborns an apgarde value greater than 8, with a
normal delivery, 48 hour stay in the hospital and no clinical
diseases diagnosed in the mother was considered normal.
The next task was to develop a similar data base for different
disease states. These were clinically diagnosed using conventional
technology to identify the disease. The patient samples were
assayed using the same protocol as the normal samples. The patients
were not transfused and the sample was less than 4 hours old at
analysis. All cell type information was collected with all enzyme
assays. Staging of the disease was noted where available and all
drug treatments were also recorded. Drug pharmokinetics were
determined by the appropriate reference to estimate the drug's
possible influence on cellular function. Separate studies using the
drugs on human tissue culture cell lines were also used to
determine their effect on specific cell types and provide
reference. Untreated patient samples were separated into their own
group and compared to drug treatment protocols. The goal of the
studies was early prediction of disease states as well as
monitoring of treatment modalities.
The data from the collected samples was organized into tables
containing rows of cell type and enzyme concentration. As
illustrated in Table 5, the first seven columns contain either
individual patients with a disease or the mean of patients with a
disease. Calculation of the sum of the squares, the covariance or
the correlation of the rows then indicated which enzyme and what
cell type gave the largest difference and therefore the most
descriptive indicator of the disease. This approach used the
Non-Negative Least Squares method and the ANOVA method.
Eigenvectors were then determined for an unknown to predict the
most likely disease for the unknown. FIG. 16 shows the prediction
of an unknown (actually diagnosed as JRA) from a group of
inflammatory diseases (Kawasaki #1, Lupus #2, Juvenile Rheumatoid
Arthritis #3, Dermatomyositis #4, Rheumatic Fever #5, and
Inflammatory Bowel Disease #6) using the full data matrix versus
reduced data matrices using respectively: the enzyme/cell type
combinations identified by eigenvector 1 alone, eigenvector 1 and 2
together, or the analysis based on squared deviations from the mean
(variance) alone.
TABLE 5
__________________________________________________________________________
LYMPHOCYTES Kawasaki's Lupus JRA Rheumatic Inflam. Ratio Ratio
Ratio Dermatomyositis Fever Bowel Dis. Eigen Eigen Inflam Dis
SUBSTRATE Dis. Lymphs. Lymphs Lymphs Ratio Lymphs Lymphs Lymphs
Vector 1 Vector 2 Variance Lymphs
__________________________________________________________________________
LEU 0.93 1.93 1.64 1.36 1.05 1.34 0.72 ALA 1.05 1.16 1.59 1.16 1.18
1.53 1.07 GLY 1.94 1.64 0.17 1.30 2.09 1.97 0.16 VK 1.19 1.25 1.60
1.39 0.74 1.08 1.29 VK-M 0.83 0.99 7.71 0.97 0.69 0.56 3 5 4.64 KA
1.53 1.93 0.10 1.39 1.19 1.75 0.07 KA-M 1.32 1.06 1.46 1.93 2.07
1.63 2.06 Z-GP 2.90 1.93 1.29 1.73 3.23 1.73 1.07 Z-GP-M 3.44 2.09
1.30 3.45 4.09 3.80 1.04 Z-TP 6.5 5.85 2.30 2.89 3.87 7.19 3.03
1.71 FDA 0.28 1.01 1.17 1.06 0.90 0.87 0.80 FDA-NAF 0.30 0.74 0.21
1.14 0.97 0.89 0.16 DCHFMESPMA 0.09 0.63 0.38 0.52 0.34 0.40 0.31
GFGA 2.15 1.25 2.03 2.14 2.14 2.16 1.40 RGES 1.43 1.84 1.43 2.06
2.04 1.96 1.23 DGLUC 0.25 0.78 0.82 0.72 0.97 1.04 0.45 DPO4 0.62
0.90 0.82 0.64 0.77 0.87 0.45 GALAC 2.46 1.30 2.28 2.34 2.24 2.39
1.40
__________________________________________________________________________
MONOCYTES Lupus JRA Rheumatic Inflam. Kawasaki's Ratio Ratio
Dermatomyositis Fever Bowel Dis. Eigen Eigen Inflam. Dis. SUBSTRATE
Dis. Monos Monos Monos Ratio Monos Monos Lymphs Vector 1 Vector 2
Variance Monos
__________________________________________________________________________
LEU 0.57 44.44 1.44 1.35 0.96 1.13 1 1 0.65 ALA 1.36 6.17 1.31 0.99
1.08 1.43 5 0.94 GLY 3.83 6.00 1.11 1.01 2.06 1.84 6 0.35 VK 5.38
1.64 5.94 1.42 1.32 3.08 7 2.32 VK-M 0.25 11.37 3.49 3.45 8.67 1.41
2 6 3 1.87 KA 2.22 6.93 0.54 1.04 2.39 1.95 4 0.25 KA-M 7.08 14.08
12.14 1.55 15.36 4.10 3 1 2 1.44 Z-GP 4.06 1.88 1.34 1.50 3.56 1.64
0.98 Z-GP-M 4.47 1.92 1.79 3.05 2.99 3.33 0.73 Z-TP 6.5 6.40 2.29
8.33 3.12 5.37 2.83 4 3.06 FDA 0.41 1.01 1.55 0.94 0.63 0.98 0.47
FDA-NAF 0.58 0.83 0.35 1.03 0.89 1.11 0.13 DCHFMESPMA 0.13 0.48
0.38 0.47 0.35 0.46 0.24 GFGA 1.77 2.18 2.29 2.01 1.36 3.42 1.67
RGES 4.65 3.03 3.98 5.22 6.52 6.02 0.61 DGLUC 0.35 0.38 1.13 0.73
2.91 4.62 0.28 DPO4 0.51 0.69 1.13 0.79 1.09 1.30 0.28 GALAC 8.40
1.17 3.81 2.14 1.80 4.93 6 1.87
__________________________________________________________________________
GRANULOCYTES Lupus JRA Rheumatic Inflam. Kawasaki's Ratio Ratio
Dermatomyositis Fever Bowel Dis. Eigen Eigen Inflam. Dis. SUBSTRATE
Dis. Grans Grans Grans Ratio Grans Grans Lymphs Vector 1 Vector 2
Variance Grans
__________________________________________________________________________
LEU 0.33 2.03 1.43 1.31 0.98 1.11 0.58 ALA 1.31 1.08 1.21 0.97 1.03
1.26 0.80 GLY 2.30 1.42 0.30 1.12 0.56 1.78 0.17 VK 1.30 1.04 1.26
1.12 0.60 1.08 0.85 VK-M 0.72 0.89 9.49 0.15 0.48 0.64 2 4 3.39 KA
0.99 1.64 0.10 1.16 1.02 1.32 0.05 KA-M 1.32 1.01 1.13 1.02 0.17
1.58 1.37 Z-GP 2.54 1.82 1.24 1.44 2.51 1.47 0.83 Z-GP-M 2.83 1.94
1.48 2.76 2.98 2.68 0.66 Z-TP 6.5 4.21 2.19 5.70 2.79 4.73 2.20 5
2.01 FDA 0.31 1.01 1.12 0.92 0.67 0.93 0.50 FDA-NAF 0.39 0.72 0.23
0.97 0.80 0.96 0.08 DCHFMESPMA 0.05 0.31 0.38 0.34 0.14 0.28 0.13
GFGA 1.93 0.10 2.26 1.85 2.10 2.78 1.77 RGES 0.36 1.36 1.01 1.62
1.54 1.60 0.76 DGLUC 0.22 0.77 0.98 1.18 0.13 1.64 0.38 DPO4 0.69
0.71 0.98 0.88 0.24 1.22 0.38 GALAC 2.32 1.01 3.42 1.76 1.16 2.35
1.37
__________________________________________________________________________
In the above Table the following abbreviations are used:
LEU--(Leu).sub.2 Rho 110
ALA--(Ala).sub.2 Rho 110
GLY--(Gly).sub.2 Rho 110
VK--(Val-Lys).sub.2 Rho 110
VK-M--(Val-Lys).sub.2 Rho 110 (modified)
KA--(Lys-Ala).sub.2 Rho 110
KA-M--(Lys-Ala).sub.2 Rho 110
Z-GP--(carbobenzyloxycarbonyl-Gly-Pro).sub.2 Rho 110
Z-GP-M--(carbobenzyloxycarbonyl-Gly-Pro).sub.2 Rho 110
(modified)
Z-TP6.5--(carbobenzyloxycarbonyl-Thr-Pro).sub.2 Rho 110 (pH
6.5)
FDA--fluorescein diacetate
FDA-NAF--fluorescein diacetate in a buffer containing sodium
fluoride
DCHFMESPMA--dichlorofluorescein in MES buffer plus phorbolmyristate
acetate
GFGA--(Gly-Phe-Gly-Ala).sub.2 Rho 110
RGES--(Arg-Gly-Glu-Ser).sub.2 Rho 110
DGLUC--(D-glucose).sub.2 fluorescein
DPO4--(PO.sub.4).sub.2 fluorescein
GALAC--(D-galactose).sub.2 fluorescein
The reagents designated as "--M" or "modified" contain cofactors,
modulators, inhibitors, etc. as shown in Table 1.
As illustrated from FIG. 16, utilizing only the eigenvector 1 and 2
or the squared deviation from the means analysis provides the
correct diagnosis, whereas eigenvector 1 alone cannot distinguish
from diseases #3 and #4. From Table 5, using three cell types and
18 enzyme concentrations, it is apparent that only six or eleven
values are necessary to classify the unknown sample. The
information most informative to the disease diagnosis came from the
monocyte cell type and cathepsin, aminopeptidase and dipeptidyl
peptidase enzymes.
EXAMPLE 34
Analysis and Diagnosis of Normal and Diseased States using
Artificial Intelligence
Artificial intelligence was used to analyze data of cellular enzyme
functions, and determine normal and disease states. Peptidases were
used to distinguish leukemia from non-leukemia (output pattern).
For input patterns lymphocyte and granulocyte cell types were used
with aminopeptidases, cathepsins and dipeptidylpeptidase enzyme
activities. In this Example, illustrated in Tables 6A-6C, three
normals and three leukemia patients were used as known output
patterns for the neural network to learn (in practice, the larger
the learning set the more accurate unknown prediction will be).
Unknowns were then presented to the learned algorithm as shown in
the test case examples. Clinical diagnosis was confirmed by
physicians. The trained neural network was able to correctly
classify the leukemia from the non-leukemia.
TABLE 6A ______________________________________ THE USE OF
PEPTIDASES TO DISTINGUISH LEUKEMIA FROM NON-LEUKEMIA
______________________________________ Aminopeptidases are Pro,
Lys, Gly, ala; Cathpsin B and Gln/Ser, Val/Ser and Leu/gly;
Cathepsin C is Thr/.Pro and Dipeptidylpeptidase IV is Gly/Pro. The
results of this study shows 100% predictability of leukemia when
tested against normal and various diseases as specified. LEARNED
CASES: Substrates: Pro, Lys, Gly, Ala, Gln/Ser, Thr/Pro, Val/Ser,
Leu/Gly and Gly/Pro Patients: ID # DIAGNOSIS**
______________________________________ 1. N191 Normal 2. N192
Normal 3. N193 Normal 4. J4 Acute Leukemia 5. J5 Acute Myelogenous
Leukemia 6. J4 Acute Leukemia
______________________________________ TEST CASES: Patients: ID #
DIAGNOSIS** ______________________________________ 1. J4 Acute
Leukemia 2. J19 Chronic Lymphocytic Leukemia 3. J22 Acute
Myelogenous Leukemia 4. N199* Normal 5. N200 Normal 6. N201 Normal
7. N206 Normal 8. P68 Abnormal - Tachycardia 9. P69 Abnormal -
Pancreatic Cancer 10. N191 Normal 11. N192 Normal 12. N193 Normal
13. J4 Acute Leukemia 14. J5 Acute Myelogenous Leukemia 15. J4
Acute Leukemia 16. P70 Abnormal - Cirrhosis/Hemobilia 17. P71
Abnormal - Acute Pyelonephritis 18. N194 Normal 19. N195 Normal 20.
N197 Normal 21. N202 Normal 22. J22 Acute Myelogenous Leukemia
______________________________________ *Normal donor later found to
have cervical cancer **Diagnosis provided by Jackson memorial
Hospital; Normal donors were inhouse employees
TABLE 6B ______________________________________ TEST CASE #4
______________________________________ Synopsis: Lymphs and Grans
were used on Leam and Test cases with the following results: SCORE
I.D. # Negative Leukemic Diagnosis
______________________________________ 1 J4 81.4 Acute Leukemia 2
J19 100 Chronic Lymphocytic Leukemia 3 J22 77.9 Acute Myelogenous
Leukemia 4 N199* 63.5 Normal 5 N200 100 Normal 6 N201 100 Normal 7
N206 100 Normal 8 P68 100 Tachycardia 9 P69 100 Pancreatic Cancer
10 N191 100 Normal 11 N192 100 Normal 12 N193 100 Normal 13 J4 100
Acute Leukemia 14 J5 99.4 Acute Myelogenous Leukemia 15 J4 99.3
Acute Leukemia 16 P70 100 Cirrhosis/Hemobilia 17 P71 100 Acute
Pyelonephritis 18 N194 95.6 Normal 19 N195 100 Normal 20 N197 99
Normal 21 N202 98.5 Normal 22 J22 72.4 Acute Myelogenous Leukemia
______________________________________ PREDICTION: 100%
Non-Leukemic Leukemic ______________________________________ Dx
Non-Leukemic 15/15 0/15 Dx Leukemic 0/7 7/7
______________________________________ *Normal donor later found to
have cervical cancer
TABLE 6C
__________________________________________________________________________
CLASSIFICATION OF NEW CASES
__________________________________________________________________________
J4 11/21/91 J5 11/27/91 P70 P71 ACUTE LEUK. A M L
CIRHOSIS/HEMOBILIA AC. PYELONEPHRITIS
__________________________________________________________________________
2.72 GP Lymphs 3.40 GP Lymphs 6.68 GP Lymphs 3.55 GP Lymphs 0.47
PRO L 0.89 PRO L 0.56 PRO L 0.52 PRO L 0.77 LYS L 5.71 LYS L 1.02
LYS L 0.91 LYS L 14.02 GLY L 28.36 GLY L 54.84 GLY L 41.93 GLY L
86.00 ALA L 92.44 ALA L 141.87 ALA L 109.26 ALA L 0.05 QS L 0.07 QS
L 0.04 QS L 0.05 QS L 2.30 TP L 1.32 TP L 1.89 TP L 1.86 TP L 0.05
VS L 0.05 VS L 0.04 VS L 0.08 VS L 0.16 LG L 0.23 LG L 0.79 LG L
0.68 LG L 10.75 GP Grans 8.95 GP Grans 16.42 GP Grans 11.07 GP
Grans 5.77 PRO G 5.92 PRO G 0.66 PRO G 6.33 PRO G 3.01 LYS G 14.86
LYS G 2.33 LYS G 4.17 LYS G 27.79 GLY G 56.33 GLY G 88.42 GLY G
59.35 GLY G 193.43 ALA G 194.85 ALA G 236.23 ALA G 185.37 ALA G
0.22 QS G 1.24 QS G 0.19 QS G 0.51 QS G 5.96 TP G 2.72 TP G 4.02 TP
G 4.37 TP G 0.37 VS G 1.13 VS G 0.26 VS G 0.69 VS G 0.85 LG G 2.51
LG G 2.20 LG G 4.00 LG G 0.00 NORMAL 0.63 NORMAL 100.00 NORMAL
100.00 NORMAL 100.00 LEUKEMIC 99.37 LEUKEMIC 0.00 LEUKEMIC 0.00
LEUKEMIC
__________________________________________________________________________
N194 N195 N197 N202 NORMAL NORMAL NORMAL NORMAL
__________________________________________________________________________
5.22 GP Lymphs 5.24 GP Lymphs 5.97 GP Lymphs 6.61 GP Lymphs 0.86
PRO L 0.88 PRO L 0.57 PRO L 0.89 PRO L 0.93 LYS L 1.07 LYS L 0.70
LYS L 0.71 LYS L 39.76 GLY L 48.18 GLY L 42.96 GLY L 44.55 GLY L
97.02 ALA L 118.81 ALA L 96.36 ALA L 118.37 ALA L 0.02 QS L 0.04 QS
L 0.06 QS L 0.01 QS L 1.82 TP L 1.90 TP L 1.96 TP L 2.49 TP L 0.07
VS L 0.06 VS L 0.11 VS L 0.04 VS L 2.06 LG L 1.93 LG L 1.79 LG L
1.28 LG L 12.49 GP Grans 13.31 GP Grans 15.85 GP Grans 14.44 GP
Grans 3.75 PRO G 4.70 PRO G 3.76 PRO G 4.96 PRO G 1.56 LYS G 2.53
LYS G 1.59 LYS G 13.22 LYS G 60.30 GLY G 79.13 GLY G 76.88 GLY G
66.22 GLY G 164.00 ALA G 221.28 ALA G 188.20 ALA G 201.17 ALA G
0.07 QS G 0.21 QS G 0.24 QS G 0.08 QS G 3.77 TP G 3.74 TP G 3.82 TP
G 4.44 TP G 0.29 VS G 0.37 VS G 0.58 VS G 0.24 VS G 3.52 LG G 4.11
LG G 4.41 LG G 2.70 LG G 95.56 NORMAL 100.00 NORMAL 99.02 NORMAL
98.62 NORMAL 4.46 LEUKEMIC 0.00 LEUKEMIC 1.00 LEUKEMIC 1.32
LEUKEMIC
__________________________________________________________________________
J22 12/9/91 J4 12/2/91 A M L A L
__________________________________________________________________________
3.01 GP Lymphs 3.80 GP Lymphs 0.44 PRO L 0.29 PRO L 1.20 LYS L 1.81
LYS L 27.14 GLY L 27.32 GLY L 83.48 ALA L 68.10 ALA L 0.02 QS L
0.01 QS L 1.02 TP L 1.46 TP L 0.04 VS L 0.03 VS L 0.61 LG L 0.18 LG
L 11.52 GP Grans 11.08 GP Grans 3.53 PRO G 3.59 PRO G 1.90 LYS G
4.09
LYS G 38.47 GLY G 47.74 GLY G 157.48 ALA G 139.53 ALA G 0.24 QS G
0.44 QS G 2.51 TP G 2.59 TP G 0.32 VS G 0.14 VS G 1.94 LG G 0.79 LG
G 27.64 NORMAL 0.31 NORMAL 72.43 LEUKEMIC 99.81 LEUKEMIC
__________________________________________________________________________
J4 12/3/91 J19 11/27/91 J22 12/2/91 N99.degree. ACUTE LEUK. C L L A
M L NORMAL
__________________________________________________________________________
4.17 GP Lymphs 1.73 GP Lymphs 1.73 GP Lymphs 5.55 GP Lymphs 0.24
PRO L 0.44 PRO L 0.40 PRO L 1.02 PRO L 1.64 LYS L 0.98 LYS L 0.74
LYS L 0.33 LYS L 31.51 GLY L 11.39 GLY L 24.50 GLY L 30.68 GLY L
74.64 ALA L 47.78 ALA L 84.42 ALA L 91.07 ALA L 0.06 QS L 0.06 QS L
0.02 QS L 0.02 QS L 1.18 TP L 0.50 TP L 0.99 TP L 1.86 TP L 0.04 VS
L 0.01 VS L 0.01 VS L 0.03 VS L 0.19 LG L 0.02 LG L 0.02 LG L 0.85
LG L 1.28 GP Grans 1.39 GP Grans 13.98 GP Grans 13.41 GP Grans 3.68
PRO G 5.50 PRO G 4.03 PRO G 4.85 PRO G 3.19 LYS G 4.41 LYS G 1.41
LYS G 0.95 LYS G 48.53 GLY G 48.80 GLY G 34.02 GLY G 45.00 GLY G
138.89 ALA G 143.11 ALA G 157.12 ALA G 157.30 ALA G 0.19 QS G 1.16
QS G 0.10 QS G 0.05 QS G 1.93 TP G 2.95 TP G 2.33 TP G 3.71 TP G
0.23 VS G 2.83 VS G 0.22 VS G 0.14 VS G 1.06 LG G 36.35 LG G 1.56
LG G 2.022 LG G 18.76 NORMAL 0.00 NORMAL 22.16 NORMAL 63.52 NORMAL
81.42 LEUKEMIC 100.00 LEUKEMIC 77.90 LEUKEMIC 36.50 LEUKEMIC
__________________________________________________________________________
N200 N201 N206 P68 NORMAL NORMAL NORMAL TACHYCARDIA
__________________________________________________________________________
5.38 GP Lymphs 6.53 GP Lymphs 7.09 GP Lymphs 5.92 GP Lymphs 0.74
PRO L 1.10 PRO L 1.28 PRO L 0.70 PRO L 0.90 LYS L 1.05 LYS L 1.65
LYS L 1.51 LYS L 42.28 GLY L 45.32 GLY L 55.56 GLY L 59.29 GLY L
101.38 ALA L 106.72 ALA L 122.71 ALA L 1471.10 ALA L 0.01 QS L 0.02
QS L 0.04 QS L 0.02 QS L 2.39 TP L 1.88 TP L 2.89 TP L 1.90 TP L
0.06 VS L 0.07 VS L 0.28 VS L 0.05 VS L 1.55 LG L 1.57 LG L 3.12 LG
L 0.78 LG L 12.74 GP Grans 15.92 GP Grans 17.96 GP Grans 16.08 GP
Grans 4.04 PRO G 6.42 PRO G 6.13 PRO G 7.19 PRO G 1.46 LYS G 2.06
LYS G 3.11 LYS G 3.09 LYS G 59.39 GLY G 75.48 GLY G 91.93 GLY G
100.00 GLY G 171.06 ALA G 206.06 ALA G 232.57 ALA G 276.07 ALA G
0.06 QS G 0.09 QS G 0.27 QS G 0.33 QS G 4.69 TP G 4.23 TP G 5.98 TP
G 4.63 TP G 0.24 VS G 0.32 VS G 1.03 VS G 0.33 VS G 3.40 LG G 3.14
LG G 6.01 LG G 2.52 LG G 100.00 NORMAL 100.00 NORMAL 100.00 NORMAL
100.00 NORMAL 0.00 LEUKEMIC 0.00 LEUKEMIC 0.00 LEUKEMIC 0.00
LEUKEMIC
__________________________________________________________________________
P69 N191 N192 N193 PANCREATIC CA NORMAL NORMAL NORMAL
__________________________________________________________________________
6.80 GP Lymphs 4.14 GP Lymphs 3.00 GP Lymphs 3.00 GP Lymphs 0.66
PRO L 0.69 PRO L 1.14 PRO L 0.95 PRO L 1.22 LYS L 1.18
LYS L 0.56 LYS L 0.52 LYS L 67.38 GLY L 43.57 GLY L 37.47 GLY L
39.30 GLY L 130.34 ALA L 95.72 ALA L 103.31 ALA L 100.50 ALA L 0.31
QS L 0.02 QS L 0.03 QS L 0.03 QS L 2.26 TP L 2.14 TP L 2.12 TP L
2.33 VS L 0.18 VS L 0.14 VS L 0.08 VS L 0.09 VS L 2.00 LG L 2.42 LG
L 3.45 LG L 1.56 LG L 16.21 GP Grans 11.20 GP Grans 11.22 GP Grans
8.87 GP Grans 5.70 PRO G 4.34 PRO G 4.34 PRO G 4.75 PRO G 2.14 LYS
G 1.20 LYS G 1.20 LYS G 1.43 LYS G 88.04 GLY G 73.20 GLY G 43.67
GLY G 63.33 GLY G 204.02 ALA G 177.32 ALA G 154.84 ALA G 178.64 ALA
G 0.37 QS G 0.19 QS G 0.15 QS G 0.14 QS G 4.21 TP G 4.73 TP G 4.62
TP G 4.96 TP G 0.57 VS G 0.64 VS G 0.40 VS G 0.44 VS G 3.53 LG G
5.37 LG G 6.78 LG G 3.65 LG G 100.00 NORMAL 100.00 NORMAL 100.00
NORMAL 98.77 NORMAL 0.00 LEUKEMIC 0.00 LEUKEMIC 0.00 LEUKEMIC 1.23
LEUKEMIC
__________________________________________________________________________
TABLE 7
__________________________________________________________________________
ABERRANT ENZYME ACTIVITIES IN 10 FEBRILE CHILDREN
__________________________________________________________________________
LYMPHS PATIENTS P88 P89 P92 P95 P96 P97 P99 P101 P102 P103
__________________________________________________________________________
1 Leu + + + + + o o + o + 2 Ala o o o o o o 3 Pro o o o 4 Lys o o o
5 Gly o - o o o o 6 Ser o o o 7 Arg o o - + o o o 8 Arg-TFA o o o 9
Asp o o o 10 Val/Ser - o o o + - - o 11 Val/Ser-M o o o 12 Val/Lys
o o o - o o + o o o 13 Val/Lys-M o o o + + + o o o 14 Gln/Ser o o o
16 Gln/Ser-M o o o 16 Leu/Gly o - - o + - o o 17 Leu/Gly-M o o o 18
Lys/Ala o o o o o o o o o 19 Lys/Ala-M o o o o o o + o o 20
Z-Ala/Ala - - - - o - o - 21 Z-Ala/Ala-M - - - 22 Z-Gly/Pro o - - -
- o - - 23 Z-Gly/Pro-M - - - - - 24 Gly/Leu o o o 25 Gly/Leu-M o o
o 26 Ala/Gly - - - - - - o o o 27 Ala/Gly-M - - - 28 Ala/Ala-TFA o
o o 29 Ala/Ala-M o o o 30 TP 6.5 - - - o o 31 TP 6.5-M - o o 32 LLR
o o o 33 LLR-M 34 LGLG o o o 35 LGLG-M 36 FDA o + + + + o o + o +
37 FDA-NaF o + + + + o o + o + 38 DCFH o + + + + o o + o + 40
DCFH.sub.-- PMA o + + + + + o + o + 42 GPLGP o o - - o - 43 GPLGP-M
- o o 44 GFGA o o o o o 45 RGES o o o o + o o o o o 46 DGLUC o o o
- 47 DPO4 o o o o - o o o o o 45 GALAC o o o o o 49 TP 8.7-M - o o
50 TP 8.7 - o o 51 DIGLUC o o o Enteritis Viral Adenitis Viral
Viral Pyelonephritis Viral Fever-Unknown UTI Viral Syndrome
Stomatitis Syndrome Syndrome Syndrome Origin
__________________________________________________________________________
MONOS PATIENTS P88 P89 P92 P95 P96 P97 P99 P101 P102 P103
__________________________________________________________________________
1 Leu + + + + + + o + o + 2 Ala o o + + + o 3 Pro o o o 4 Lys + o +
5 Gly o o o + o 6 Ser o o + 7 Arg + o o o o o + 8 Arg-TFA + o + 9
Asp o o o 10 Val/Ser - - o - + - - o 11 Val/Ser-M - - o 12 Val/Lys
- - o o o o + o - + 13 Val/Lys-M - o - o o o - - o 14 Gln/Ser o o +
16 Gln/Ser-M - - o 16 Leu/Gly o - o o + o o 17 Leu/Gly-M - o o 18
Lys/Ala o o o o o + o + o o 19 Lys/Ala-M - - - - - - o o - 20
Z-Ala/Ala - - o o o - o o 21 Z-Ala/Ala-M o o o 22 Z-Gly/Pro o o - o
o o o - o o 23 Z-Gly/Pro-M - o - o o 24 Gly/Leu o o o 25 Gly/Leu-M
- o o 26 Ala/Gly - - o - o o o o o 27 Ala/Gly-M - o o 28
Ala/Ala-TFA + + + 29 Ala/Ala-M - o o 30 TP 6.5 - - - o o 31 TP
6.5-M - o o 32 LLR o o + 33 LLR-M 34 LGLG o o o 35 LGLG-M - - + 36
FDA o + + + + + o + o + 37 FDA-NaF o + + + + o + + + 38 DCFH o + +
+ + + o + o + 40 DCFH.sub.-- PMA o + + + + + o + o + 42 GPLGP - o -
- - 43 GPLGP-M o + + 44 GFGA o o o o o 45 RGES - o + o o o o - o o
46 DGLUC - o - + 47 DPO4 o + + + o o + o + 45 GALAC o o o o o 49 TP
8.7-M - o o 50 TP 8.7 - o o 51 DIGLUC + o + Enteritis Viral
Adenitis Viral Viral Pyelonephritis Viral Fever-Unknown UTI Viral
Syndrome Stomatitis Syndrome Syndrome Syndrome Origin
__________________________________________________________________________
GRANS PATIENTS P88 P89 P92 P95 P96 P97 P99 P101 P102 P103
__________________________________________________________________________
1 Leu + + + + + o o + o + 2 Ala o o o o o o 3 Pro o o o 4 Lys + + o
5 Gly o o o o o o 6 Ser + o o 7 Arg + o o + + o + 8 Arg-TFA + + + 9
Asp o o o 10 Val/Ser - - o o + - o 11 Val/Ser-M o o o 12 Val/Lys -
- o - o + + o o o 13 Val/Lys-M - o o + + o o - o 14 Gln/Ser + o +
16 Gln/Ser-M + o o 16 Leu/Gly o - o o + o o - 17 Leu/Gly-M - - - 18
Lys/Ala o o o o o o o + o o 19 Lys/Ala-M o o o o o o o o o 20
Z-Ala/Ala - - o o o o o 21 Z-Ala/Ala-M o o o 22 Z-Gly/Pro o - - o o
o o 23 Z-Gly/Pro-M - - o o 24 Gly/Leu o + o 25 Gly/Leu-M o o o 26
Ala/Gly + o o o o o o o o 27 Ala/Gly-M - o o 28 Ala/Ala-TFA + + +
29 Ala/Ala-M o o o 30 TP 6.5 - - - o 31 TP 6.5-M - o o 32 LLR o o o
33 LLR-M 34 LGLG o o o 35 LGLG-M o o o 36 FDA o + + + + + o + o +
37 FDA-NaF o + + + + o + + o + 38 DCFH o + + + + + o + o + 40
DCFH.sub.-- PMA o + + + + + o + o + 42 GPLGP o o - - o - 43 GPLGP-M
- o o 44 GFGA o o o o o 45 RGES o + + o o o o o o o 46 DGLUC o + +
+ 47 DPO4 o + + o o o o + o + 45 GALAC o o o o o 49 TP 8.7-M - o o
50 TP 8.7 o o o 51 DIGLUC o o o Enteritis Viral Adenitis Viral
Viral Pyelonephritis Viral Fever-Unknown UTI Viral Syndrome
Stomatitis Syndrome Syndrome Syndrome Origin
__________________________________________________________________________
+ >0.25 Increase over normal range - >0.25 Decrease over
normal range o No change
The use of neural networks has also been used to reduce a large
data set (3 cell types x 45 enzyme assays x 5 or more diseases) to
only the important cell types and enzyme assays for classification
of a disease. This can be visualized by graphing the ratio of the
diseases to the mean of the normal of all patients with the
disease, as illustrated in FIGS. 17A, 17B and 17C to reduce the
data set, as illustrated in Table 5, and providing an HLA score
sheet, illustrated in Table 7, showing greater than .+-.25% of the
mean normal enzyme activity for that cell type.
EXAMPLE 35
Determination of Disease Progression Using Artificial Intelligence
Based Analysis of Cellular Enzyme Function
Progression of disease during treatment and monitoring a return to
normalcy during treatment are shown in FIGS. 18A-18F and the raw
data is summarized in FIG. 18G. The time measurements were
monitored by three-dimensional plotting of cell-type enzyme
activity patterns of the sample values and normal values. FIG. 18A
illustrates raw data for lymphocyte cells of a particular patient
as compared to the normal data. The raw data in FIG. 18A was taken
on August 25. FIG. 18B illustrates the raw data of the diseased
patient as compared with normal data on September 6. Similarly,
FIGS. 18C-18F illustrate raw data for the diseased patient as
compared to normal data on September 21, September 26, October 12,
and October 13, respectively. The time progression illustrated in
FIGS. 18A-18F clearly indicates by October 13, the raw data of the
"diseased patient" is now virtually identical to the normal data.
The increase in stage or complication with additional disease
states may also be performed in a manner similar to that
illlustrated in FIGS. 18A-18F. All patents and publications
referred to in this application are hereby incorporated by
reference in their entirety.
The invention has been described with reference to the preferred
embodiments. It should be understood, however, that the invention
is not so limited, and the scope of the invention should be
determined with reference to the following claims, rather than to
the foregoing specification.
* * * * *